Optogenetic Brain Interfaces
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Pashaie, Ramin, Polina Anikeeva, Jin Hyung Lee, Rohit Prakash,
Ofer Yizhar, Matthias Prigge, Divya Chander, Thomas J.
Richner, and Justin Williams. “Optogenetic Brain Interfaces.”
IEEE Rev. Biomed. Eng. 7 (2014): 3–30.
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http://dx.doi.org/10.1109/RBME.2013.2294796
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Optogenetic Brain Interfaces
Ramin Pashaie*, Polina Anikeeva*, Jin Hyung Lee*, Rohit Prakash*, Ofer Yizhar*, Matthias Prigge*, Divya
Chander*, Thomas Richner*, Justin Williams*
Abstract—The brain is a large network of interconnected
neurons where each cell functions as a nonlinear processing
element. Unraveling the mysteries of information processing in
the complex networks of the brain requires versatile neurostimulation and imaging techniques. Optogenetics is a new stimulation
method which allows the activity of neurons to be modulated by
light. For this purpose, the cell-types of interest are genetically
targeted to produce light-sensitive proteins. Once these proteins
are expressed, neural activity can be controlled by exposing the
cells to light of appropriate wavelengths. Optogenetics provides
a unique combination of features, including multi-modal control
over neural function and genetic targeting of specific celltypes. Together, these versatile features combine to a powerful
experimental approach, suitable for the study of the circuitry of
psychiatric and neurological disorders.
The advent of optogenetics was followed by extensive research
aimed to produce new lines of light-sensitive proteins and to
develop new technologies: for example, to control the distribution
of light inside the brain tissue or to combine optogenetics with
other modalities including electrophysiology, electrocorticography, nonlinear microscopy, and functional magnetic resonance
imaging.
In this paper, the authors review some of the recent advances
in the field of optogenetics and related technologies and provide
their vision for the future of the field.
Index Terms—Optogenetics, Brain Interface, Micro-ECoG,
Optrode, 2-photon Neurostimulation, Optogenetic fMRI.
I. I NTRODUCTION
Over the last few decades, the dominant hypothesis in
treating neurological and psychiatric diseases has been the
chemical imbalance paradigm which assumes any mental
disorder is the result of imbalance in the concentration of
chemicals in the central or peripheral nervous system. Based
on this hypothesis, such diseases are curable if we invent
mechanisms to control and monitor the concentration of the
* All authors have contributed equally. Corresponding should be sent to:
Ramin Pashaie, e-mail: pashaie@uwm.edu.
Ramin Pashaie is with the Electrical Engineering Department, University
of Wisconsin-Milwaukee, 3200 N Cramer St., Milwaukee, WI, 53211, e-mail:
pashaie@uwm.edu.
Polina Anikeeva is with the Material Sciences and Engineering Department,
Massachusetts Institute of Technology, 77 Mass. Ave., Cambridge, MA 02139,
anikeeva@mit.edu.
Jin Hyung Lee is with the Bioengineering, Neurology and Neurological
Sciences, and Neurosurgery Departments, Stanford University, CA 94305,
USA, e-mail: ljinhy@stanford.edu.
Rohit Prakash is with the Bioengineering Department and Medical school
of Stanford University, CA, 94305, ropra@stanford.edu.
Ofer Yizhar and Matthias Prigge are with the Department of Neurobiology, Weizmann Institute of Science, Rehovot, 76100, Israel, e-mails:
ofer.yizhar@weizmann.ac.il, matthias.prigge@weizmann.ac.il
Divya Chander is with the medical school of Stanford University, CA,
94305, e-mail: dchander@stanford.edu
Justin Williams and Thomas Richner are with the Biomedical Engineering Department, University of Wisconsin-Madison, WI 53706, e-mails:
williams@engr.wisc.edu, richner@wisc.edu
Manuscript received —-;
corresponding chemicals by using appropriate pharmacological substances. This approach has been somewhat successful
in treating several mental disorders such as depression or
anhedonia that are caused by the reduction in the concentration
of the monoamine neurotransmitter serotonin [1].
Recent advances in the development of brain interface
instrumentation and prosthetic devices has opened a new line
of research to treat mental disease by speaking the electrical
language of neurons, usually known as interventional psychiatry. Deep-brain stimulation (DBS) [2], [3] is an example of
this approach which has been reasonably successful in treating
some neurological diseases including Parkinson’s. Nonetheless, most interventional therapeutic procedures lack specificcell-type targeting capabilities and potentially cause serious
side effects. For instance, implanted electrodes in Parkinsonian
patients stimulate most cells in their reach with no preference
to target any specific cell-type, and as a result, cause several
side effects including depression, mood alteration, or sensory
and motor control problems which are all suppressed by
turning off the stimulations pulses.
To address this challenge, a new neurostimulation technique,
known as optogenetics, was invented by combining the tools
of molecular genetics with recent advances in the fields of
optics and photonics. In this technique, a family of lightgated microbial opsin proteins that function as light-activated
proteins, are expressed in genetically targeted neurons [4], [5],
[6], [7], [8], [9], [11], [12]. Once these light sensitive proteins
are expressed in a neuron, the activity of the cell can be
increased or suppressed, with millisecond temporal accuracy,
by exposing the cell to light with appropriate wavelengths,
even without the addition of the exogeneous cofactor in
mammalian cells.
Some major advantages of optogenetic stimulation are:
- Specific cell-type targeting can be achieved in optogenetics
by controlling the gene delivery process to express light
sensitive proteins predominantly in the cell-type of interest.
This goal can be achieve, for example, by using appropriate
promoters [13].
- Bidirectional control of cellular activities is feasible in
optogenetics. It is possible to simultaneously express cation
channels and anion pumps that are sensitive to different
wavelengths in one cell-type of interest. Thus, by exposing
the cell to appropriate wavelengths, researchers can depolarize
or hyperpolarize the neurons to manipulate their activities [5],
[14].
- The inherent parallelism of optics can help to manipulate
neural activities in large-scale neural networks of the brain,
particularly in the cortex.
Some major applications of optogenetic neuromodulation
are:
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-Cracking neural codes: Neurons communicate by generating sequences of action potentials while information is
embedded in the mean firing rate or the precise timing of
action potentials. As described earlier, optogenetic tools provide a bidirectional mechanism to control neural activities with
millisecond temporal resolution. Consequently, by engineering
a sequence of light pulses, any firing pattern is producible
which, in principle, represents a specific neural code. Then,
with a suitable quantitative readout, the effect of the generated
codeword on the post-synaptic neurons or the corresponding
behavioral phenotypes can be investigated,
- Interrogating neural circuits: By providing a discriminative mechanism for controlling the activity of cell-types in a
neural network, optogenetics enables the dissection of mental
disease circuitries (e.g., Parkinsonian neural circuits [15]) or
interrogation of the role of circuit elements in the overall
dynamics of the network (e.g., functionality of fast-spiking
Parvalbumin inhibitory interneurons in cortical microcircuits
of prefrontal cortex [16]),
- Generating reversible models of neurological diseases:
The mechanism for bidirectional control of genetically designated neural populations gives the opportunity to emulate and
study new models of neurological diseases (e.g., optogenetics
can be used to reversibly generate patterns of epileptogenic
activity [4]),
- Developing new and efficient therapeutic treatments: Optical modulation of activity in targeted cell-types can help in
the discovery of efficient treatments for mental diseases with
minimum side effects, since we can selectively modulate the
activity of excitatory or inhibitory neurons separately (e.g.,
hyperpolarization of glutamatergic neurons in subthalamic
nucleus (STN) with expression of NpHR and suitable light
exposure has shown to be effective in treating Parkinson’s [15]
in animal models of the disease).
The advent of optogenetics has opened new lines of research to develop other light sensitive ion channels with faster
or slower kinetics or different spectral sensitivities. It also
opens new fields of investigation to control the distribution
of light inside the three-dimensional structure of the brain
tissue or combine optogenetics with other stimulation or imaging modalities including electrophysiology, electrocorticography, nonlinear microscopy, and functional magnetic resonance
imaging.
In this paper, the authors review some of the recent advances
in the field of optogenetics and related technologies and
provide their vision for the future of the field. In the section
II, the progress in development of new tools of optogenetics
via bioprospecting search or genetic engineering is discussed.
Mechanisms of light delivery are explained in section III,
while section IV is devoted to probes that are designed
and microfabricated for optogentic stimulation. The recently
developed methodologies to combine optogenetics with fMRI
are discussed in section V. Multi-photon stimulation is another
new development in the field which allows researchers to reach
deeper within the cortex and simultaneously stimulate and
image neural activities. Multi-photon stimulation is covered in
section VI. An example of a hybrid brain interface platform
where optogenetic stimulation is combined with electrocor-
2
ticography (ECoG) is detailed in section VII and applications
of optogenetics in the study and treatment of neurological diseases are covered in section VIII. Finally, concluding remarks
are summarized in section IX.
II. T OOLS OF O PTOGENETICS AND M ECHANISMS OF
G ENE D ELIVERY
A. Light-activated proteins
Optogenetics applies light-sensitive proteins which have
been isolated from various microorganisms and plants, to manipulate excitable cells in heterologous systems. Initial work in
the field used naturally occurring photosensitive proteins such
as channelrhodopsin (ChR) [6] and halorhodopsin (HR) [5] to
induce activation or inhibition of neural activity in mammalian
neurons and this has opened up a rapidly expanding field
utilizing additional genetically encoded actuators in a wide
range of applications [7], [8], [9]. Beyond the utilization of
such naturally-occurring photoreceptors, protein engineering
over the last decade has generated an expanded optogenetic
toolbox for more precise and effective manipulation, allowing
refined modes of control that are tailored to individual systems.
The most widely used optogenetic tools are proteins of the
microbial rhodopsin family [10]. These proteins were first
discovered in the 1970s and were already then thought to
serve as a ”scientific goldmine” for renewable energy [17].
Microbial rhodopsins are single-component units, which can
transform light energy into current with efficiency higher than
any man-made mechanisms so far. Microbial rhodopsins such
as bacteriorhodopsin (BR) and halorhodopsin, were found in
the archaea Halobacterium salinarium which inhabit extreme
environmental surroundings such as the Dead Sea in Israel
[18], [19]. In these ancient halobacteria, rhodopsins are believed to use light energy for generating the electrochemical
gradient required for ATP synthesis in the absence of oxygen
and for withstanding the high osmotic pressure in these
extreme surroundings [20]. In 2002, a new member of the
microbial rhodposin family was isolated from the fresh water
algae Chlamydomonas rheinhardtii [21]. Unlike previously
discovered microbial rhodopsins, which typically serve as ion
pumps and transport ions against electrochemical gradients,
channelrhodopsins (ChRs) possess an aqueous ion channel
pore, allowing passive conduction of cations across the cell
membrane, according to the electrochemical gradient [22].
Therefore, microbial rhodopsins can be grouped into pumps,
acting as photodiodes, and light-triggered ion channels which
can be considered as light-dependent resistors. Regardless of
the mechanism of action or the ion species conducted, all
microbial rhodopsins rely on the same fundamental photoreaction comprising the isomerization of a covalently bound retinal
co-factor, Figure 1(a). Upon photon absorption, the co-factor
isomerizes from so called all-trans to 13-cis conformation.
This conformational shift changes the dipole moment of the
retinal molecule and initiates pronounced structural rearrangements of the protein, which finally lead to the transport of
ions [23]. All these complex rearrangement occur within less
than a millisecond whereby the activated rhodopsin cycles
through various photochemically distinct states. This series of
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Fig. 1. Photocycle of the wild-type ChR2 and light-induced photocurrents. (A) Schematic drawing of channel opening upon light illumination. In the closed,
dark state the light antenna, all-trans retinal, can isomerize after absorption of a photon to 13-cis retinal, thereby triggering opening of the channel. (B) The
light-induced changes in the electrical properties of the channels upon opening can be modeled as a 4-state photocycle consisting of two dark states (D1 and
D2) and two light-induced open states O1 and O2. (C) Illustrates the electrical response of a single cell expressing ChR2 to two successive light pulses. Upon
illumination a transient peak inward current is triggered, which decays to the stationary photocurrent. This drop in photocurrent is commonly referred as
inactivation. Upon light-off the photocurrent returns to baseline with an apparent τ of f 21ms. The seconds light pulse only evokes a smaller peak photocurrent
while reaching the same stationary current. The time it takes to return to full peak current size is known as recovery time. (D) Shows current-clamp recordings
of neurons expressing NpHR (top) and ChR2 (bottom). Action potentials can be triggered with pulses of blue light in ChR2-expressing cells (473nm) whereas
spontaneous activity can be suppressed by activating eNpHR3.0 with yellow light (589nm).
states can be collectively described as a photocycle in various
level of detail. Figure 1(b) shows a 4-state electrophysiological
photocycle, which explains the major electrical features of
channel function, including inactivation, photocurrent kinetics
and adaption phenomena as observed in electrical recordings
from cells expressing channelrhodopsin (Figure 1(c)) [24]. For
example, a fully dark adapted ChR2 molecule activated with a
short blue light pulse will first proceed through an open state of
high conductance (O1), causing a transient peak photocurrent
and then entering a lower conductive state (O2) which leads
to lower photocurrent. This drop in photocurrent is commonly
referred as inactivation, Figure 1(c) and can be as large as
72% of the initial current for the wild-type ChR2 [25]. The
dwell time for the conductive states is estimated to be 10ms
[26]. Molecules in the open state can transition to two different
dark states with an apparent τ1,2 of f off of 21ms [26], [25].
Upon repeated illumination at this stage, molecules re-excited
from the second dark state will then only pass through the low
conductance state, giving rise to a light-adapted steady-state
photocurrent which is smaller than the initial peak current,
Figure 1(c). Consequently, a series of 2-ms light pulses will
cause attenuation of the photocurrent to a lower equilibrium
level [25]. Only after a recovery period of > 6s all molecules
will have returned to their initial dark-adapted state, Figure
1(c).
To achieve light-based control of neural function, these
bio-electric units have been selectively targeted to different
excitable cells such as neurons, heart or muscles cells [6],
[27], [28], [29]. In neurons, microbial pumps such as HR
or BR induce a square-like hyperpolarization response of
the membrane potential upon illumination [5], [30], which
can rapidly and effectively silence neuronal activity, Figure
1(d)(upper trace). ChRs perform the opposite function of
depolarizing neurons in response to illumination by allowing
positively charged cations to passively enter the cell. ChRs
therefore serve as light-induced activators. Figure 1(d)(lower
trace) shows a pulsed activation of neurons expressing ChR2.
Since light naturally penetrates intact biological tissues, the
optogenetic approach has been extended to living organisms,
including nematodes, flies, zebrafish, rodents and monkeys
[28], [31], [32], [33], [34].
Driven by the success of the initial approach with microbial rhodopsins isolated from several species, large genomic
screens for new microbial rhodopsins in all kingdoms of life
have led to the discovery of several new variants, which
show diverse functional properties such as spectral tuning,
ionic selectivity and compatibility towards mammalian cell
expression [35], [36], [37], [38]. These proteins, encoded
by genes from diverse microbial species, were subjected to
IEEE REVIEWS IN BIOMEDICAL ENGINEERING, DRAFT-REVISED
molecular engineering which aimed to increase their efficacy
as light-driven tools for controlling neural excitability. Generally, two lines of improvement strategies have been applied.
The first was aimed toward increasing the biocompatibility
to mammalian cells. The second approach was to modify
the intrinsic properties of the actuator along one or more
functional dimensions. Optimization of membrane targeting
was mainly required for the inhibitory microbial rhodopsins,
most of which were derived from prokaryotic species in which
membrane proteins are produced and transported using mechanisms significantly different from that used in mammalian
cells. To improve the proper assembly of these rhodopsins,
signaling peptides, which act as a intercellular ”zip code”,
from endogenous ion channels have been added to each end
of the microbial rhodopsin [39]. The addition of Golgi- and ER
export signals from mammalian ion channels reduced protein
aggregation and increased the number of rhodopsin molecules
reaching the plasma membrane [12]. This strategy turned out
to generalize quite well, and many available actuators used in
optogenetic research these days make use of such sequences
[40]. As a consequence, the most commonly used tools for
neural silencing are the two enhanced versions: NpHR3.0 and
Arch3.0 [14].
Whereas the main improvements for hyperpolarising tools
were focused on increasing photocurrent amplitude through
enhanced membrane targeting, ChR engineering has been very
versatile over the last few years. Molecular engineering of ChR
variants has yielded numerous tools with distinct spectral and
kinetic properties. Two mutations, H134R and T159C, have
been shown to increase photocurrent amplitude in pyramidal
neurons by a factor of 1.5 to 2 [28], [41]. In both variants,
this change corresponds with slower off-kinetics of 30-60ms
in neurons corresponding to a prolonged dwell time [26], [14],
[25]. One of the key amino acids controlling the protonation
state of the retinal chromophore is a glutamate at position 123
in the ChR2 sequence. Substitution of an alanine or threonine
at this position generated the E123A/T mutants, in which the
photocycle is accelerated to an apparent off-kinetics of 46ms [42]. These mutations can increase the fidelity of high
frequency evoked action potentials trains. However, since the
open state is populated only for shorter times, the amount
of ions conducted during this interval is decreased. This is
effectively manifested in reduced photocurrent amplitude. For
the same reason, the necessary light intensity to reach half
saturation is increased from 1mW/mm2 to 5mW/mm2 [14],
placing constraints over in-vivo applications in which tissue
heating could in fact pose a significant challenge [43].
To address the heating issue and to increase the effective
light sensitivity of optogenetic excitation, light switchable
variants have been engineered. Mutations at positions C128
and D156 can substantially delay the closing reaction of the
pore. The single mutations C128S and D156A slow down the
apparent off-kinetics of ChR2 to 120s and 250s, respectively
[44]. Combining both mutations generated a variant with
a closing time constant of 30 minutes. Spectroscopic data
revealed that, as with the wild-type ChR2 [45], yellow light
can facilitate the closing reaction [46]. Hence, these mutants
can be triggered into the open state by a brief blue light
4
pulse and can then be flashed back to the closed, dark state
with intense yellow light. Due to the off-on nature of the
light-driven changes in membrane potential, these tools were
named step function opsins (SFOs; [44]). Since activated SFOs
accumulate in the open state for the entire duration of blue
light illumination, even low light intensities will render all
molecules active when pulse duration is prolonged. Therefore,
those variants act as photon integrators and are thus 100
times more light-sensitive than naturally occurring microbial
rhodopsins [9]. Long low-intensity light pulses can also reduce
the variability of activation throughout the targeted cell population in-vivo since the full population of opsin-expressing
cells, even in a large volume of tissue, could depolarize over
time [46].
With respect to ion selectivity, under physiological ionic
conditions and membrane resting potential of a neuron, the
photocurrent of ChRs is composed out of 65% H+, 35% Na+
and 1% Ca2+ and Mg2+ [47]. Only few mutations have been
shown to have a significant impact on ion selectivity. Most
notably the L132C mutation in ChR2, which was found to
increase conductance of the divalent cations Ca2+ and Mg2+
by a factor of five [47]. The ChR2 L132C/T159C mutants
show large photocurrents with an increased conductance of
divalent cations. Since Ca2+ can trigger intracellular signaling
cascades, these variants can potentially be used to manipulate
cell activity by changing intracellular ionic composition. Such
alterations to the ionic selectivity of ChRs can potentially be
very useful, if selectivity can be manipulated to the extent that
only single ions (e.g. sodium, potassium or chloride) can be
exclusively conducted. Although this is probably a difficult
task, it is likely that the recently resolved three dimensional
crystal structure of ChR [48] will further boost this type of
molecular development.
Following the success of applying microbial rhodopsins as
optogenetic tools in mammalian neurons, similar approaches
have been applied to engineer a range of light-driven actuators
that allow the manipulation of diverse cellular processes. One
common approach involves addition or substitution of enzyme
domains with photoreceptor units, which exhibit structural
changes upon illumination. For this purpose, the most commonly used photoreceptor domains are LOV (light-oxygenvoltage domain), BLUF (blue light sensor using FAD), cryptochromes and phyotchromes. These photoreceptors all bindto
organic co-factors such as flavins and different porphyrin
molecules which essentially perform a similar role to that
played by the retinal chromophore in the rhodopsins [49].
Here, most notable is a photoactivatable adenylate cyclase
bPac, which was found in the bacteria Beggiatoa sp. Habitas
hydrogen sulfide rich environments. This cyclase is lightsensitive via a BLUF domain. In Drosophila, bPac expression
increases the level of signaling molecule cAMP by a factor of
10 upon blue light illumination [50]. Recently, the toolbox was
extended by a very promising member - a light-inducible transcriptional effector (LITE) for endogenous gene expression.
Here a light inducible dimerization of a cryptochrome called
Cry2 with its native truncated binding partner CIBN [51], was
fused to a transcription effector domain and a DNA recognition
domain, respectively. Upon blue light illumination the DNA
IEEE REVIEWS IN BIOMEDICAL ENGINEERING, DRAFT-REVISED
bound CIBN recruits the Cry2-fused activator domain and can
thereby increase the transcription of target genes [52]. These
and other tools have extended the range of optogenetic manipulation beyond the simple control of neuronal excitability and
are likely to contribute significantly to a range of experimental
applications.
Despite the increased number of applications, all present
optogenetic tools utilize light in the visible range. New tools,
sensitive at the longer range of the electromagnetic spectrum
(e.g. infrared or radio frequency), are desirable since they
would allow deeper tissue penetration and perhaps even facilitate non-invasive approaches. Promising candidates for this
approach, for example, are the infrared thermal sensitive ion
channels from bats and phythochrome-based tools [53], [54].
Tools which can be specifically switched on and off with
minimal cross-excitation would allow consecutive control of
different cellular processes without optically cross-activating
each other in the same organism.
General speaking ChRs impose another activation pathway
on the top of naturally occurring ones. Upon light stimulation,
action potentials can be evoked in an all-or-none fashion.
From an analytical point of view, genetically-encoded tools
rendering specific endogenous lightactivatable proteins would
be more valuable, since they would enable researchers to
modulate the endogenous cellular mechanisms which drive
actions potentials. But the greatest need for improvements
in the field of optogenetics are the development of cellular
activity reporters. Particularly, fast, non-invasive reporters for
membrane potential at the far end of the electromagnetic spectrum would be very valuable. In addition, effective optogenetic
reporters at different wavelength for Ca2+, cAMP, cGCMP or
different neuromodulators such as dopamine or acetylcholine
are still needed or have room for improvement. Together with
the growing range of optogenetic actuators, these tools will
allow the characterization of brain dynamics on levels other
than electrical activity.
Manipulating cellular processes with light at high spatialtemporal resolution has already been proven as an indispensable method in neuroscience research. Therefore, the
preparadigmatic stage, where optogenetic tools are developed
just in a proof-of-concept fashion has passed. With the increasing knowledge of the mechanisms of action of these proteins
and the complexities of their application in neural systems,
new tools should be engineered such that they maximally fulfill
their intended function and allow predictable, well-controlled
manipulation of neural activity.
B. Gene delivery considerations in optogenetics
One of the most important strengths of optogenetic methodology is the fact that the actuators, microbial rhodopsins or
other proteins discussed above, are genetically-encoded. This
allows scientists to target specific cell populations for lightbased manipulation. These populations can be defined based
on genetic signatures called promoters, with DNA sequences
that are selectively activated only in particular circuits or cell
types. By coupling the microbial opsin gene to a promoter
of choice and inserting this DNA into neurons, expression of
5
the opsin will only occur in cells that selectively express the
chosen promoter. Therefore, application of optogenetics invivo requires the genetic modification of neurons to induce
expression of the light-gated tools. Although many methods
exist for such gene delivery [55], genetically engineered
viruses [56] are by far the most popular means of delivering
optogenetic tools. Lentiviral vectors (LV) [57], [58] and adenoassociated viral vectors (AAV) [59] have been widely used
to introduce opsin genes into mouse, rat, and primate neural
tissues [8]. These vectors allow high expression levels over
long periods of time with little adverse effects [59]. AAVbased expression vectors are less immunogenic, and some
types of AAV-based vectors allow the transduction of larger
tissue volumes compared with LV due to their small particle
size and increased viral titers. They are therefore increasingly
used both in basic research and in gene therapy trials [60].
Additionally, AAV is considered safer than LV as the currently
available strains do not broadly integrate into the host genome
and are thus rated as biosafety level (BSL) 1 or 2 agents.
Both LV and AAV vectors can be used in conjunction with
cell type-specific promoters ([58], [61], [62], facilitating their
application in cell-type specific optogenetic manipulation.
III. M ECHANISMS OF L IGHT D ELIVERY
To study brain microcircuits via optogenetics, we need
light delivery mechanisms to control the distribution of light
not only on the surface but also inside the brain tissue.
Distribution of light on the surface can be controlled precisely
by spatial light modulators (SLMs). To deliver light inside
the tissue, lightguides, e.g. optical fibers, are implanted in the
brain to guide laser pulses with appropriate wavelengths to
regions of interest. Recently, more advanced mechanisms such
as micro-fabricated channel waveguide arrays or holographic
microscopy systems are being developed and tested to control
the distribution of light even in the three-dimensional structure
of the brain.
A. Waveguiding Systems
Optical fibers are the most popular lightguides that are
used to stimulate deep brain objects. In most applications,
an aspheric lens couples the beam of a solid-state laser to
a multimode step index fiber which guides and deliver the
optical power to the region of interest. Superluminescence
light emitting diodes (LEDs), which are more stable light
sources, are also becoming popular for optogenetic stimulation. Electronically driven LEDs are particularly suitable for
integrated systems such as prosthetic devices. However, coupling efficiency dramatically drops when incoherent sources
are used and as a result, researchers usually increase the
fiber diameter to couple enough light into the fiber. The
stereotaxic surgery protocols for implanting fibers are currently
well established [63], [11], Figure 2(a), (b). Obviously, optical
fibers only deliver light to the area close to the fiber tip.
To control the distribution of light in the three-dimensional
structure of the brain, more complex light delivery mechanisms
are needed.
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6
Fig. 2. (A) Optical fiber implanted in the brain tissue to deliver light and stimulate deep brain objects in transfected areas. (B) A rat receiving two implants
in the motor cortex on both hemispheres. (D) Structure of a multichannel probe [64], (D) and multichannel waveguide arrays [65]. (E) A side-firing fiber
that radiates power almost orthogonal to the optical axis of the fiber [66]. (F), (G) Microactuator assembly that moves a side-firing fiber inside the brain;
mechanical design and a prototype [66].
It is possible to make an array of channel waveguides where
each waveguide in the array terminates at a different depth
[64]. An example of this approach is displayed in Figure
2(c). Here, a 1.45cm-long probe is microfabricated in the form
of a 360 micron-wide array of 12 parallel silicon oxynitride
(SiON ) multimode waveguides clad with SiO2 and coated
with aluminum. Each waveguide in the array accepts light
from a light source at its input terminal and guides the coupled
light down to an aluminum corner mirror which deflects light
away from the probe axis. Based on the published data, light
losses are relatively small so that each waveguide can almost
deliver about 30% of the injected light at the output terminal.
The main benefits of this approach are the simplicity of the
design and efficiency of light delivery. Moreover, light pulses
can be delivered independently at multiple sites following any
arbitrary temporal sequence of interest. A new generation of
such probes have been recently developed and tested where a
matrix of waveguide arrays are integrated on a single substrate
to build a prosthetic device which can potentially control the
light distribution in different layers of cortex [65], Figure 2(d).
The major drawback of this approach is the overall size of the
probe, which is relatively large, and the complexity of the
optics required to couple light into the channel waveguides in
large arrays.
Another more simple approach for developing a distributed
light delivery mechanism is to physically move an optical fiber
inside the tissue [66]. In this design, a microactuator assembly,
which sits on the skull, moves a side-firing fiber inside a glass
made capillary that is implanted inside the brain prior to the
experiment, Figure 2(e)-(g). Side firing fibers are optical fibers
where the tip is precisely angled and polished so that the
radiation pattern of the fiber is almost orthogonal to the optical
axis of the fiber. It is possible to optimize the parameters
of the side-firing fiber, including the tip angle and numerical
aperture, to minimize the divergence of the radiating beam and
improve the spatial resolution of the device for light delivery.
This system can deliver different wavelengths at numerous
positions inside the tissue, and the size of the capillary can
be less than 50 micron. The device has spatial resolution of
a few microns over a dynamic range of about 4mm and the
total weight of the device is less than 2g. The major drawbacks
of this design are the limited speed of the system to change
the position of the fiber. Also, it is practically difficult, if not
impossible, to integrate an array of such side-firing fibers and
microactuators into a single prosthetic device.
Another approach remained to be explored in the future is
the use of tilted fiber gratings that couple guided modes of an
optical fiber to radiation modes [67].
B. Spatial Light Modulator Based Systems
Two types of spatial light modulators are used to produce
distributed patterns of light in the brain: microelectromechanical (MEMS) based SLMs and liquid crystals.
The most well-known MEMS light modulator is the Texas
Instrument’s digital micromirror device (DMD). The DMD
system is a light processor consisting of a large array of
bistable micro-mirrors. The position of each mirror in the array
is under independent computer control. Exposure is controlled
by programming the duration each mirror reflects light toward
a sample [68]. An example of the optical design of a DMD
system which is specifically designed for optogenetic applications is shown in Figure 3(a). In most DMD designs, a highpower incoherent light source, with a wide spectrum, produces
the optical power which is filtered to select the appropriate
range of wavelengths. The filtered beam is then homogenized
by an integrating rod and collimated by telecentric optics to
expose the active area of the DMD through a total internal
reflection (TIR) prism. The TIR prism, which is placed in front
of the DMD, guides the beam of light to illuminate the DMD
surface with the right angle considering the specific design of
micro-mirrors in DMD chips. This prism also separates the
illuminating beam from the modulated reflected beam. Next,
the DMD modulates the beam and a telecentric lens projects
the pattern on the entrance pupil of an image combiner. The
image combiner is an assembly of mirrors and epi-fluorescence
optics which makes it possible to simultaneously project a
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IV. E NGINEERING N EURAL P ROBES FOR O PTOGENETIC
E XPERIMENTS
Fig. 3.
Spatial light modulator systems for optogenetic stimulation, (a)
Basic optical design of a DMD based system for optogenetic stimulation and
imaging, (b) Liquid crystal based light delivery mechanism and holographic
microscopy.
pattern on the sample and perform bright field or fluorescence
imaging.
An interesting example of optogenetic stimulation by a
DMD system is the the study of the motor circuit in freely
moving Caenorhabditis-elegans [69]. Since the DMD can
produce rapidly changing high-resolution illumination patterns, this system has been used combined with a real-time
image processing software to track a freely moving worm and
optically target its motor system with single cell resolution.
Another application of DMD based system for optogenetic
stimulation is discussed later in the discussion of optogenetic
electrocorticography recording.
Liquid crystal SLMs are specifically designed to be used
with lasers and coherent light sources. A typical design of
such systems is shown in Figure 3(b). In this design, a laser
source produces a collimated beam of light which is phase
modulated by a liquid crystal SLM and the modulated beam
in launched into the optical path of a fluorescence microscope
[70], [71]. The SLM generates a dynamic mask which reshapes
the beam of a laser to control the distribution of light on the
sample. From Fourier optics we know that a lens is nothing
but a simple phase mask. Therefore, it is possible to emulate
the function of any arbitrary lens by a phase modulating
liquid crystal to focus the beam of laser at different depths,
even in parallel, to produce three-dimensional patterns of light
distribution, such as holograms, inside the tissue [71]. Recently, liquid crystals have been integrated into the optical path
of multiphoton microscopes, and femto-second pulsed lasers
are used to produce three-dimensional patterns of optogenetic
stimulation over the entire depth of the cortex in small rodents.
In other words, dynamically changing holograms are produced
in the cortex to generate complex spatial-temporal patterns of
activities in the cortical microcircuits. This topic is discussed
further in section IV.
From the origins of modern neuroscience direct electronic
recording of neural activity has been proven essential to
detailed understanding of the dynamics and structure of neural
circuits underlying observed behaviors. Consequently, neural probe engineering has evolved into a mature discipline
covering a variety of aspects ranging from the electronic
design and materials development to tissue characterization
and computerized neural data analysis, to name a few.
Since optogenetic approaches to neural control have spread
through the neuroscience community, the designers of neural
recording devices found themselves confronted with a demand
for integration of optical elements into the neural probes.
As the majority of currently available technologies for simultaneous electrophysiology and optogenetics are based on
previously developed recording-only devices, we will review
the progress and the challenges in neural probe design and
consider the implications of opto-electronic integration at the
leading edge of neural engineering.
Over the course of the past several decades a number
of neural probe designs have been proposed, fabricated and
applied in basic as well as clinical neuroscience experiments
[72]. In this article we will focus on invasive tissue-penetrating
probes that allow for recordings of individual action potentials
(spikes) from the neighboring neurons (single units) based on
the local changes in ionic concentrations in the vicinity of
the electrodes. While these devices are primarily employed in
research, the recent applications in brain-machine interfaces
(BMI) demonstrate the utility of single-unit recordings from
motor cortex in neuroprosthetics for paralyzed patients ([73],
[74]).
In addition to single-unit recording these devices can provide the temporal profiles of local field potentials (LFPs) in
the neighboring networks by simply changing the signal filter
settings (from v300-8000 Hz for single units to v1-1000 Hz
for LFPs). LFP data can, for example, be subject to Fourier
analysis to reveal predominant oscillation frequencies within
the monitored neuronal network. Changes in LFP frequency
spectra are often characteristic to a particular neurological
disorder (e.g. Parkinson’s disease or epilepsy).
While patch-clamp electrophysiology offers the most precise recording of voltage changes across neuronal membranes,
this cell-penetrating technique suffers from relatively low
throughput and is not compatible with chronic implantation.
The vast majority of neuroscience experiments, require recordings of activity of large numbers of individual neurons during
a specific behavioral paradigm and hence single-neuron resolution and recording stability over a course of an experiment
(days to years) are often the key requirements for the neural
probe design [80].
Currently, in addition to the anatomy of the specific region
of the nervous system and the desired resolution of the
acquired data, the limitations of the available fabrication approaches dictate the neural probe geometry. Individual metallic
microwires v50-100 micron in diameter are the simplest forms
of neural probes, Fig. 4(A). These electrodes are primarily
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Fig. 4. Examples of commonly used single unit and LFP recording devices and their optogenetics-compatible modifications. (A) Silica-encapsulated
tungsten electrodes (Thomas Recording GmbH). (B) Tungsten microwire array (Tucker-Davis Technologies Inc.) (C) Tetrode microwire bundle (University of
Queensland). (D) Silicon ”Utah” multielectrode array (Blackrock Inc.). (E) Silicon multitrode probes (Neuronexus Inc.) (F) An optrode consisting of a silica
fiber and a tungsten electrode [13]. (G) Metal microwire array equipped with a silica fiber [92] (H) Optetrode combining 4 tetrode bundles and an optical
fiber (Anikeeva 2011). The inset shows the cross section schematic. (I) Utah array incorporating a tapered silica fiber [94], [101]. (J) Silicon multitrode probes
integrated with tapered silica fibers [102].
based on tungsten, steel, gold, platinum and iridium and can
be assembled into arrays of up to several tens to increase the
number of recorded single units, Figure 4(B). The ability to
distinguish between action potential shapes originating from
different neurons was shown to be improved by assembling
individual microwires into tightly wound stereotrodes (two
intertwined microwires) and tetrodes (four microwires) [75],
[76], Figure 4(C). These neural probes usually consist of
polymer-coated nickel-chromium alloy electrodes v12 micron
in diameter, since larger and stiffer electrodes do not lend
themselves to the assembly process and produce bulky and
tissue-damaging devices. Stereotrodes and tetrodes were originally developed for high-throughput recordings from dense
hippocampal layers in rodents, which remains the main application of these devices to date. These wire assemblies are often
used in combination with micro-drives that allow individual
propagation of each assembly independently thus enabling
active search of firing neurons.
Due to the maturity of the processing methods enabled by
electronics industry, silicon has become a popular material for
the fabrication of neural probes. While numerous silicon-based
designs have been produced over the past two decades their basic structure can be approximately divided into multi-electrode
”Utah” arrays [78], [77], MEA Figure 4(D) and multitrodes
[80], [82], which are sometimes referred to as ”Michigan
probes”, Figure 4(E). The former structures consist of arrays
of highly-doped silicon ”peaks” created by a combination of
direct micromachining and wet etching and insulated from
each other by regions of glass. The bodies of the electrodes
are coated with a chemical-vapor deposited (CVD) polymer
parylene C and the conductive tips are exposed. The size of the
individual electrodes within the array is on the order of several
tens of microns at the recording tip but 300-400 micron at the
base, which yields a comparatively large footprint for arrays
comprised of more than sixteen electrodes (e.g. a 10×10 MEA
has dimensions of approximately 4×4 mm2 , not including
connector parts). One of the main advantages of these devices
lies in their floating design. The MEAs are deposited on the
cortical surface attached to the connector mounted on the
skull with long flexible loosely bundled wires. As a result
these devices require a sensitive implantation procedure during
which the MEA is deployed pneumatically into the cortex.
Since rodents lack sufficient intracranial space, floating MEAs
are limited to applications in primates. Furthermore, the length
of the electrodes is restricted to v 1-1.5mm by the fabrication
and implantation procedures and hence these devices are used
primarily for cortical recordings in primates, facilitating an
entire field of brain-machine interfaces (BMI) and, recently,
neural control of robotic aids by paralyzed human patients
[73].
Multitrode probes employ a thin silicon substrate with
lithographically patterned metal electrodes distributed along
the shaft. This streamlined geometry and deterministic positioning of the recording sites makes these probes attractive
for recordings in deep brain structures with high cell density
such as the rodent hippocampus. These probes have recently
been extended to devices incorporating logical elements thus
enabling on-site signal amplification, which holds the potential
to improve the yield of recorded single units. However, the
lengths of the silicon shaft is still limited to a few millimeters
by the micromachining process restricting the applications to
experiments in rodents and cortical recordings in primates.
Similarly to stereotrodes and tetrodes these devices can be
combined with microdrives. However, due to the device geometry, the entire shaft is propagated through the brain thus
advancing all of the patterned recording channels.
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Neurotrophic electrodes comprised of a silica pipette with
several internal electrodes incorporate neural tissue, which
promotes neurite ingrowth into the cone allow for high signalto-noise recordings. However, to our knowledge they are yet to
be combined with optogenetics and hence will not be discussed
in detail in this review. Interested readers may refer to [81]
for further details.
The majority of opsins, with the exception of step-function
opsins, at average expression levels (2-8 weeks of incubation
time following viral delivery into the neural tissue) require
light power densities of approximately 1 mW/mm2 in order
to robustly evoke or inhibit action potentials in-vivo. This
poses a requirement of direct light delivery into the area of
interest, as the neural tissues are highly scattering and absorptive within the visible range, which currently encompasses the
activation/inactivation spectra of all retinal-containing opsins.
Consequently the vast majority of optogenetic experiments
are performed with implanted optical fibers and the light is
supplied by an external source such as a laser or a highpower light-emitting diode (LEDs). The direct implantation
of miniature LEDs into the brain is an alternative strategy
for light delivery, which will be discussed in the following
paragraphs.
While the initial proof-of-concept optogenetic experiments
performed in-vitro employed intracellular patch-clamp recordings [6], the need for combined light delivery and extracellular
electrophysiological readout was recognized as this technology
transitioned in-vivo. Furthermore, as the state of the awake
brain of behaving rodents differs dramatically from activity
patterns of the unconscious brain of anesthetized animals, not
to mention dissociated neuronal culture a, it became essential
to observe the electrophysiological outcomes of optogenetic
stimulation and correlate them to an observed behavior. As optical fibers are routinely employed in optogenetic experiments,
their integration with electrophysiological probes provides the
most straightforward engineering strategy. Thus, to date, the
majority of commonly used neural probes have been modified
to carry externally attached conventional commercially available silica fibers.
The simplest devices ”optrodes” comprise of individual
metal electrodes and optical fibers held together by an adhesive, Figure 4(F). These devices are primarily employed
for recording of single-unit and population (combined signals
from multiple units) activity in anesthetized animals [13].
Similarly, an optical fiber can be attached to a metal electrode
array and the resulting devices are employed during behavioral experiments primarily for population activity and LFP
recordings, Figure 4(G) [92].
Single-unit recording during optical stimulation can be
readily achieved with ”optetrodes”, which comprise of an
optical fiber surrounded by several tetrode bundles, Figure
4(H) [93]. These devices allow for recording during free
behavior and provide access to multiple recording sites due to
a miniature concentric mechanical drive. While these devices
insure illumination of all the neurons that can be recorded with
the tetrodes they do not allow for independent manipulation
of individual tetrodes.
In order to introduce a fiber into a Utah-style MEA one
9
of the electrode positions can be replaced with an opening,
Figure 4(I) [94]. In this case the scattering within the tissue
only allows for illumination of cells within the direct vicinity
of the fiber. Consequently, the utility of this approach is mainly
in exploring the effects of localized stimulation or inhibition on
a broader cell population that can be accessed by electrodes
distributed in a several millimeter area, as only a few (v49) electrodes will be in contact with the illuminated tissue.
The integration of a fiber with a Utah MEA compromises the
otherwise floating design of the device, as the conventional
silica fibers are too rigid and, hence, anchor the device to the
skull.
Silicon multitrode probes can be outfitted with fibers similarly to optrodes by simply attaching the latter to the former
with an adhesive following the fabrication process, Figure
4(J) [102]. These devices often employ tapered fibers, which
allow for reduction of the overall device dimensions but
significantly increase optical losses and reduce the numerical
aperture leading to reduced illuminated tissue volume. The
relative position of the fiber with respect to the electrode
pads determines, which of the channels will record activity
of the illuminated neurons. Positioning the fiber at the top
of the array may require very high light powers to reach the
electrodes at the tip of the array, while positioning of the fiber
tip within the array pads compromises the recording on the
upper pads as the tissue may be damaged or pushed away by
a relatively large fiber.
Connector design remains one of the main challenges for
simultaneous neural recording during optogenetic experiments
as optical coupling is required in addition to an electrical
connector. This imposes the design constrains, which often
lead to devices too large or heavy to be employed during
free behavior in smaller mammals (mice, bats) or birds. High
fidelity optical coupling (approaching 100% transmission) can
now be achieved with knife-edge polish of pigtailed ferrules
(stainless steel or zirconia) held together with ceramic sleeves.
While ferrule to ferrule connection is not considered stable by
photonics standards, its performance is sufficient for behavioral experiments on timescales of minutes to hours. Ferrule
coupling, however, restricts the position of the fiber with
respect to the recording device, which generally results in a
fiber illuminating only a fraction of the recording electrodes.
Note, that all the devices discussed so far are based on
rigid materials (metals, silicon, silica) with Young’s moduli
(e.g., E(Si)=130-170 GPa, E(SiO2)=73 GPa, E(W)=411 GPa,
E(Pt)=168 GPa) that are many orders of magnitude higher
than that of neural tissues (E v kPa-MPa) [79], [83]. It is
thought that this mismatch in stiffness contributes to tissue
damage and subsequent decay of the recording quality [84],
[85]. It is reasonable to assume that the insertion of the
implant into the tissue causes neuronal death and displacement,
however, the initial inflammatory response usually ceases
within approximately 2 weeks following the implantation,
which yields improved signal-to-noise ratio (SNR) and the
overall number of single units recorded. However, the SNR
deteriorates and the number of monitored units decays over
the course of following weeks to months [97]. While the
origins of neural probe failure are being actively investigated,
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Fig. 5. (A) Contact printed AlInGaP LED on a PDMS substrate [87]. (B)
Releasable GaN micro-LED for deep-structure optogenetics [90].
several complimentary hypotheses have been proposed. During
normal movement, the brain undergoes several tens of microns
shifts (micromotion) with respect to the skull [98], [99].
Consequently, the tissue surrounding the hard fixed implant
is subject to chronic damage, which is manifested in glial
and astrocytic activity resulting in device encapsulation. If
the latter were a dominant mechanism of device failure,
the floating Utah arrays would be immune to failure, which
is not the case. The disruption of glial networks by the
implants significantly exceeding the dimensions of individual
cells has also been suggested as a potential trigger of glial
and astrocytic response. This is supported by the reduced
glial scarring around miniature implants with cross sections
approaching single cell size. Finally, recent work suggests
that the disruption of blood-brain barrier (BBB) is yet another
factor associated with the tissue response to the implanted
devices [82]. The latter findings imply that the sharper devices
such as Utah MEA or multitrode probes produce a greater
BBB damage and yield more severe gliosis as compared to the
metal electrode arrays consisting of relatively blunt cylindrical
wires [95].
As the majority of optogentic neural probes incorporate
rigid silica fibers of 50-400 micron in diameter, it is reasonable to expect that the reliability of the neural probes
will be further compromised by such opto-electronic integration. Furthermore, so far this review has focused on the
devices designed for applications within the brain, while the
recent interest in the peripheral nervous system as a potential
early therapeutic target for optogenetics emphasizes the need
for devices tailored to highly mobile and flexible nerves.
Consequently, flexible, biocompatible platforms are needed
for the advancement of combined long-term optogenetic and
electrophysiological studies.
Over the past several years a number of technologies have
spurred in attempt to address the elastic modulus mismatch
between neural probes and neural tissues. Silicone resins
(predominantly poly(dimethyl sulfoxane), PDMS) have been
successfully employed as substrates for metal electrode arrays
by Lacour and Fawcett among others [96], [100]. This allowed
for the development of chronically implanted peripheral nerve
cuffs capable of single-unit recordings from the nerve surface.
This technology however, has not yet been integrated with
light delivery, as silica fibers are rigid, and hence an alternative
solution such as an LED array is required. Furthermore, this
technology is not yet compatible with applications beyond the
10
nerve or cortical surface (micro-ECoG) as it has limited resolution due to the fabrication constraints and the implantation
surgery into the deep tissue is yet to be developed.
Similarly, parylene C has been employed as a substrate for
thermal molded soft cone-electrodes incorporating recording
sites within the cone. These devices are potentially intended
to operate akin to neurotrophic silica-cone electrodes and may
yield improved SNR and stability. However this technology is
not currently scalable to high-density recordings, nor does it
lend itself to the incorporation of optical fibers [86] thus facing
the challenges of the silicone-backed devices.
Micro-contact printing process developed by Rogers and
colleagues has been recently employed in fabrication of sophisticated integrated structures on highly flexible silicone,
polyimide and silk fibroin substrates, Figure 5(A) [87], [88],
[89]. This innovative approach takes advantage of the mature
chip-scale semiconductor processing and combines it with
designer pre-strained interconnects, which enable the release
and transfer of the devices fabricated on wafers onto flexible
substrates. Consequently, these devices can be designed to
integrate a variety of electronic components enabling on-site
data processing as well as micro LEDs based on proven
group III-V semiconductors. The utility of these devices lies
primarily in the cortical LFP recordings as the resolution of
the printing process limits the channel size. The devices, integrating a single recording channel with LEDs, photodetectors
and thermal sensor (resistor) have been recently tested in
deep brain structures of mice, Figure 5(B). The insertion into
the depth of the tissue in this case was enabled by silicon
microneedles adhered to the devices by resorbable silk fibroin
layers [90]. However, the mechanics of the printing process
would yield comparatively large structures (several millimeters
in area) if the number of recording sites were to increase to
several tens or hundreds, hence these structures are currently
limited to a small number of electrodes.
A more traditional approach to flexible neural probes borrows fabrication strategies developed by micro-electro mechanical systems (MEMS) industry and produces devices
resembling multitrode probes, in which silicon substrates have
been replaced with polyimide [91]. In this case optogenetics
can be enabled by integrated polymer waveguides based on
photoresists poly(methyl methacrylate) (PMMA) or SU-8,
Figure 6. The resulting structures are very flexible and require
temporally stiffening encapsulation in order to penetrate deep
into the tissue. A number of encapsulation strategies such as
coatings with sugars, poly(ethylene glycol) (PEG) and, more
recently, tyrosine-based polymers allow to tune the time of
the implant softening into the neural tissue. One the main
challenges of this approach is relatively low transmittance of
the polymer waveguides (>1 dB/cm), which translates into the
need for high input light powers (>100 mW sources) in order
to enable opsin-facilitated optical neural interrogation at the
tip of fairly short (length < 5 mm) polymer waveguides.
Currently, MEMS-style fabrication and the micro-contact
printing processes suffer from the relatively low yield, which
drives the high cost of these devices reducing the number of
animals that can be employed in a given optogentic behavioral
study and hereby increasing the variability of the results.
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The design and implementation of future intimate and integrated optogenetic electrophysiological probes now depends
on the collaboration between materials scientists, chemists,
electronics, photonics and packaging engineers as the solutions
to the opto-electronic integration challenges would also need
to address the biocompatibility and reliability issues stemming
from the fragile and inhomogeneous structure of the neural
tissue.
Fig. 6. A photograph (A) and a tip close-up (B) of a MEMS-processed
polyimide-backed device equipped with a SU-8 waveguide and drug delivery
channel [91].
V. O PTOGENETIC S TIMULATION C OMBINED WITH
M AGNETIC R ESONANCE I MAGING
Furthermore, the wafer-scale processing limits the device
dimensions to the applications in rodents and the primate
cortex. Applications such as deep brain areas or the peripheral
nerves in primates or humans currently remain inaccessible
with these state-of-the-art technologies for combined optical
stimulation and recording.
A robust fabrication approach is needed to enable integration of optical components (LEDs and/or waveguides) and
high-density single-unit electrophysiology. The development
of such integrated devices may require exploration of new
materials systems such as conductive polymers and polymer composites, which have been shown to improve the
recording SNR and stability. Electrospinning and chemical
vapor deposition (CVD) enable processing of polymer probes
with diameters as low as 10 micron [103], [105]. However,
the application of these methods to integrated multi-channel
devices will require further development as the number of
materials investigated to date remains limited.
Fabrication methods allowing for processing polymers beyond photoresists may allow to reduce the losses in flexible
waveguides. The photonics industry may provide insights into
high-throughput fabrication of high-quality optical waveguides. For example, thermal drawing process, routinely used
to produce conventional silica fibers, can actually be applied
to processing of multiple materials such as polymers, metals
and semiconductors [104]. Initial attempts to employ thermal
drawing in the design of combined optical and electrical neural
probes demonstrate the promise of this approach, however, the
scaling to high-density electrophysiology as well as combined
optical and electronic connectorization would need to be
addressed before this fabrication method can be adopted at
large.
Nanowires of III-IV semiconductors provide highly intimate
interfaces with individual neurons and can also be designed
to incorporate p-n junctions necessary for LEDs and fieldeffect transistors enabling high SNR recordings [106], [107].
The majority of nanowire devices are designed on flat rigid
substrates and are primarily used for studies of individual
cultured cells. However, flexible substrates have recently enabled integration of nanowire electronics with hydrogel scaffolds creating three-dimensional hybrids of neural tissues with
embedded electrophysiological probes [108]. This approach
provides a particularly promising strategy for optogentic neural
regeneration devices, granted that challenges associated with
scalability and connectorization are overcome.
The optogenetic functional magnetic resonance imaging
(ofMRI) [109], [110], [111] technology combines optogenetic
control with high-field fMRI readout. This enables cellular
level precision in control while whole brain network function
can be readout in-vivo. Systematic control of the brain with
specificity in the cellular level combined with whole brain
dynamic imaging has been a fundamental tool missing in the
neuroimaging and neuroscience fields; and arguably has been
a key missing tool to link findings from the more precise
cellular level investigation to the overall brain function. While
standard fMRI provided important clues and insights into brain
function including mapping of visual, sensory, motor areas,
and association of brain activation changes to neurological
diseases, they were limited in fidelity due to the brain’s densely
interconnected network of heterogeneous cellular populations.
OfMRI addresses this problem by combining genetically targeted control of neurons with a method capable of whole brain
functional readout.
In the first study, it was shown that cell types specified
by genetic identity, cell body location, and axonal projection
target can be specifically excited while the whole brain function associated with the stimulation can be monitored with
spatiotemporal precision. In this study, it was first shown
that ofMRI could reliably detect local activities resulting
from cell type-specific control of neurons. For example, upon
stimulation of the ChR2-expressing excitatory neurons in
the motor cortex, a positive blood oxygen level-dependent
(BOLD) signal was observed at the site of stimulation. In
addition, the temporal shape of the optogenetically induced
BOLD responses matched the conventional BOLD signals that
are typically evoked during sensory stimulation, suggesting
that excitation of a single population is sufficient to drive the
conventional BOLD response.
Then, to demonstrate the global imaging capabilities of
fMRI, the authors measured the BOLD response at the thalamus, which has direct axonal projections from the motor
cortex. At the thalamus, robust BOLD signal was measured,
demonstrating the global imaging capability. However, an
unexpected difference in the BOLD response shape was also
observed. While largely similar in shape as the motor cortex
hemodynamic response function (HRF), there was a marked
reduction in HRF signal rise slope. To identify the source
of such difference, electrophysiology recordings were made
at the motor cortex stimulation site and the location within
the thalamus where the ofMRI signal was observed. The
electrophysiological recording revealed a delay in spike-rate
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Fig. 7. OfMRI with stimulation of excitatory neurons in the primary motor cortex. (A), During the ofMRI scan, the subject is intubated and lightly anesthetized.
The respiration rate, body temperature, and expiratory CO2 are monitored and controlled. A surface coil (not shown) is mounted and centered over the region
of interest on the skull to maximize the signal-to-noise ratio. The subject’s eyes are also covered (not shown) to avoid unintended visual stimulus. (B), Six
cycles of 20s-on-40s-off optical stimulation are delivered to the primary motor cortex of the animal during the scan. The gradient-recalled-echo (GRE) BOLD
sequence is used and 23 slices are collected for full brain coverage with 3 s temporal resolution. (C), BOLD signals are detected at both the stimulation site
(primary motor cortex, image 1,2) and downstream areas (thalamus, image 3,4). The bottom tip of the white triangle indicates the optical simulation site.
increase in thalamic neurons compared to cortical neurons,
matching the smaller HRF slope observed with ofMRI. This
opened up a possibility for the ofMRI technology to enable
not only spatially resolved neural activity mapping but also to
temporally resolve underlying activity patterns.
Next, to further expand the ofMRI technology’s capability
to map responses associated with specific modulations, the
authors visualized the brain’s response to light stimulation
delivered to the thalamus with anterograde viral injection in
M1 cortex. With anterograde vital injection in M1 cortex,
thalamic regions that receive direct axonal projection will
have opsin expression in the axons of neurons projecting
from M1. With the light stimulation of these axons, robust
activities were observation at both the thalamus and motor
cortex, demonstrating the feasibility of mapping the causal
influence of cells defined not only by genetic identity and cell
body location, but also by connection topology. Finally, the
authors showed that ofMRI could be used to trace the different
global responses to activation of two distinct thalamic nuclei
that matched predictions from literature, demonstrating the
feasibility of the in-vivo circuit function mapping capability
of ofMRI. These findings laid the basic foundation for how
the ofMRI technology can be a potent tool that can enable
systematic in-vivo circuit analysis.
Since the original publication, several studies have built
upon these results, further demonstrating the utility of the
ofMRI technology. For example, ofMRI implementation was
demonstrated in awake mice [112] that showed that a larger
BOLD response was evoked in awake animals compared
to anesthetized conditions during optical stimulation of the
primary somatosensory cortex. The study also showed that
the degree of functional connectivity between active regions
was strengthened. Therefore, under the following conditions,
ofMRI studies could preferably be performed without anesthesia: animal does not experience significant levels of stress
due to restraint and noise and importantly, large motion is that
is not compatible with the imaging setup is not evoked by the
stimulation.
The ofMRI technology have also been demonstrated at
a number of different stimulation locations including motor
and sensory cortex [109], [112], [113], ventral tegmental area
[114], and hippocampus [115], further demonstrating its utility.
Looking forward, demonstrating the ofMRI technology’s capability to identify dynamic network function across multiple
synapses also be a key next step.
Advances of the optogenetics technology [8] will also
enable diverse ofMRI experiments to be performed. For example, with optogenetics now becoming feasible in non-human
primates [61], [116], future ofMRI experiments may turn to
monkeys as an animal model for studying more complex brain
circuits that is difficult to study with rodents. Improvements
in optogenetics may also enable precise control of subcellular
compartments. With such a capability, ofMRI could provide
a unique bridge between subcellular perturbation and the
resulting global brain function. This could provide a new
platform to study the specific role of axons and dendrites in
the context of overall brain function, which have been implicated in many neurological diseases and disorders including
Alzheimer’s disease [117], multiple sclerosis [118], Autism
[119].
In addition to advances in ofMRI’s capability that is expected to come with the development of new optogenetic
tools, the development of advanced imaging technologies is
of critical importance to accelerate scientific discovery. For
example, while fMRI uniquely provides the readout of the
global brain function, the image quality is often a concern.
To accurately register neural activity to anatomical locations,
it is necessary to have high quality, distortion-free images.
Solutions to this problem include the passband SSFP fMRI
[120] method that was demonstrated in the original ofMRI
publication. Distortion-free passband SSFP fMRI combined
with optogenetics was shown to result in more robust, and
spatially accurate registration of the activities.
Another important technical development that needs to
take place is to increase spatiotemporal resolution of ofMRI
images. While fMRI is unlikely to deliver single cell level,
millisecond temporal resolution, it is quite possible that advance imaging technology development would enable corti-
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cal layer and sub-nuclei specific imaging. Furthermore, the
temporal resolution of the imaging could be improved to a
sub-second range. Note however that, for the improvement
of the spatiotemporal resolution, it is important to maintain
the whole brain volume coverage. In MRI, temporal and
spatial resolution can be easily traded off with smaller spatial volume coverage. Therefore, without the large volume
coverage, high spatiotemporal resolution is of limited value.
For example, taking several slices or even single slice images
at high spatiotemporal resolution, might be of interest for a
dedicated study looking at that particular spatial location, it
does not allow the key advantage of ofMRI, which is the
global imaging capability, to be fully utilized. A method that
can fundamentally improve spatial and temporal resolution
without sacrificing the volume coverage therefore is of critical
importance.
With the large degree of freedom in neuronal cell population
control including the type of cell targeted, the temporal pattern
of stimulation, and the type of response elicited (e.g. excitation
or inhibition), methods for robust, fast, and high-throughput
data acquisition in a closed-loop system will greatly accelerate
scientific discovery utilizing the ofMRI technology. As a first
step in achieving a real-time ofMRI system, Fang et al. [121]
developed a highly parallelized method for performing image
reconstruction, motion correction, and activation detection in
a fraction of the typical MRI acquisition repetition time.
Combined with methods such as compressed sensing [122] and
multiple coil arrays [123], cryo-cooled coils will for signalto-noise ratio (SNR) improvement, real-time image acquisition
and activation detection can be achieved. With such a system
is in place, it will be possible to interactively perturb specific
brain circuits and immediately visualize the network-level
activity that results. The simplest form of closed-loop ofMRI
would rely on manual inspection of the evoked patterns of
brain activity and corresponding change of stimulation parameters. To enable a more robust and high-throughput system,
automated analysis techniques that can identify activations and
quantify their spatial and temporal characteristics could also
be combined. Integration of such automated analysis methods
with real-time image acquisition will considerably accelerate
the discoveries made with ofMRI technology.
Optogenetic fMRI enables the study the causal global effect
of perturbing specific brain circuit elements in live animals.
This has great potential to providing insights into the mechanisms that underlie normal and diseased brain function, and
driving the development of new therapies. For example, ofMRI
may be used to track how the functional interactions within the
brain progress over time. Specifically, ofMRI may be used to
identify which brain regions fail to communicate with another
region properly, how they fail, and when. Studies have shown
that global structural and functional changes can predate clinical symptoms of neurological diseases [124], [125] and ofMRI
will provide an important new opportunity to systematically
assess changes during a disease’s progression. Unlike standard
neuroimaging techniques, ofMRI can attribute these changes
to the modulation of selective neuronal elements and temporal
parameters of stimulation (i.e. temporal encoding). This will
illustrate the highly dynamic nature of brain circuits [110],
13
and provide information about the manifestation of a disease
not obtainable with other techniques.
Just as ofMRI can be used to visualize how neurological
diseases affect the brain network function, it can also be used
to determine how a diseased brain will respond to therapy.
For example, optogenetic control may be used to selectively
simulate separate elements of neuromodulation therapeutic
targets currently in use in humans, such as in deep brain
stimulation and spinal cord stimulation therapies. Monitoring
how the brain responds to different parameters of stimulation
with ofMRI could then be used to predict which parameters
optimize therapeutic benefit.
Given the extensive library of literature that has used
fMRI to study neurological diseases, a subtle yet significant
contribution of ofMRI experiments to the clinical domain
will also come from an improved understanding of the blood
oxygenation level dependent (BOLD) responses. Despite two
decades of fMRI research in humans, the nature of the
BOLD signal remains poorly understood, due to an incomplete
understanding of the relationship between neural activity and
haemodynamic processes in the brain [126], [127], [128].
Optogenetic fMRI provides a unique opportunity to probe
these systems and to determine the role that different neuronal
populations play in generating these responses. The original
ofMRI paper [109] was the first to demonstrate this principle,
showing that causal activation of a specific single population
of neurons could lead to BOLD responses with classical
kinetics. Since then, a number of other ofMRI experiments
have begun to disentangle the convoluted relationship between
neural activity and the BOLD signal. For example, one study
compared the temporal characteristics of the ofMRI BOLD
response with different electrophysiological measurements,
finding that it correlated best with neuronal spiking activity
[129]. Another study showed that the BOLD signal summates
in response to closely spaced trains of stimulation, confirming
that the BOLD signal provides a linear approximation of
neural activity levels [113].
To further link the BOLD response evoked by optical
stimulation with the fMRI responses typically measured during
sensory or cognitive tasks, quantitative comparison of the
two will also be important. It has already been shown that
optical activation of ChR2-expressing neurons can lead to local
changes in blood flow while the normal activation of neurons
through glutamatergic receptors would be blocked under the
same condition [130]. Future integration utilizing combinatorial optogenetics with ofMRI will allow the contributions of
distinct cell populations to the BOLD signal to be even further
dissected. In current ofMRI experiments, the optically driven
population likely recruits or inhibits other neuronal types and
glial cells in generating the measured fMRI signal. With
multiple wavelengths of light control used to prevent additional
populations from being excited or inhibited, resulting signal
would reflect the change solely caused by the initially targeted
population. This information could be used to interpret fMRI
results by identifying the types of neuronal elements involved.
A simple example of this includes understanding the effect of
suppressing neural activity compared to activating inhibitory
neurons. Since the BOLD response only provides an indirect
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14
Fig. 8. Schematic of a high-throughput, closed-loop ofMRI system. fMRI images are acquired using ultrafast acquisition techniques during optogenetic
stimulation in awake or anesthetized animals. High-throughput, real-time image reconstruction, motion correction, and activation detection are performed in
series. The resulting patterns of activity can by analyzed with automated algorithms or inspected manually to decide how to update stimulation parameters.
Degrees of freedom available include the location and type of cell targeted, the temporal pattern of stimulation, and the type of modulation (e.g. excitation
versus inhibition). (Modified Figure modified from [121]).
measure of neural activity, without a proper tool to control the
neural elements with precision while measuring fMRI signal,
how these two distinct processes which both reduce the level of
neural activity affect the measured signal has remained elusive.
The significance of ofMRI lies in its ability to map global
patterns of brain activity that result from the selective control
of precise neuronal populations. Currently, no other existing
method can bridge this gap between whole-brain dynamics and
the activity of genetically, spatially, and topologically defined
neurons. While a number of studies have already started to
exploit the unique capabilities of this technology, ofMRI will
likely experience significant growth and development over the
next decade. Both the advances in the molecular toolbox of
optogenetics, and improvements in imaging technology hold
important roles in brining ofMRI closer to its full potential.
In particular, the integration of ultra-fast, high-resolution data
acquisition and analysis, and combinatorial optogenetics will
enable powerful closed-loop ofMRI that can real-time visualize brain activity with real-time feedback control. Further
research uncovering the nature of the BOLD signal will also
make it possible to extract more detailed information about the
neural activity measured in fMRI studies. Finally, the application of ofMRI to translational research has the potential to
fundamentally transform therapeutics design for neurological
disorders. New targets of therapeutic neuromodulation will
likely be discovered, and improved models of neurological disease will be introduced. Taken together, ofMRI is anticipated
to play an important role in the future understanding of both
normal and diseased brain functions and enable alternative
treatment developments.
VI. C OMBINING O PTOGENETICS WITH T WO -P HOTON
M ICROSCOPY
As discussed earlier, methods for guiding spatial delivery of
single-photon light excitation have been used to improve the
precision of optogenetic modulation [69], [131] - [138]. Light
localization with higher precision can be achieved using multiphoton excitation microscopy. In neuroscience, two-photon
excitation microscopy has allowed for the direct investigation
of the nervous system at different length scales (from synapses
to neural circuits) and on different time scales (milliseconds
to months) [139], [140]. The physiological importance of such
work is highlighted in studies investigating the role of the
hippocampus in spatial navigation tasks in rodents. Dombeck
and colleagues used two-photon microscopy in combination
with genetically encoded calcium indicators in the mouse
hippocampus to map the spatial representation of place cells
and found that the anatomical proximity of neurons had no
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15
Fig. 9. Two-photon optogenetic control of spike firing in-vivo in adult mammals, a. Schematic of the experimental setup for in-vivo two-photon control
of layer 2/3 somatosensory neurons transduced with C1V1T-p2A-YFP, b. Left, two-photon image of layer 2/3 pyramidal neurons transduced with C1V1Tp2A-YFP in somatosensory cortex (150-250 micron below pia). Right, two-photon image of layer 1 pyramidal neurons transduced with C1V1T-p2A-YFP in
somatosensory cortex (50-150 micron below pia), c. Two-photon image of dendritic spines on pyramidal cells transduced with C1V1T-p2A-YFP in layer 2/3
of somatosensory cortex, d. Upper left, in-vivo two-photon image of layer 2/3 pyramidal cells transduced with C1V1T-p2A-YFP (imaged under loose patch).
Lower left, trace showing precise spike train control with 5Hz 1040nm raster-scanning illumination. Upper right, axial resolution of two-photon optogenetic
control of spiking in-vivo. Blue triangles indicate pyramidal neurons and red boxes illustrate ROI positioning. Spiking of layer 2/3 cells as a function of raster
scan position is shown. Lower left, lateral resolution of two-photon optogenetic control of spiking in-vivo.
correlation to the spatial place field they represent [141]. Such
observations necessitate the formation of tools that enable
researchers to take forward control of the activity of single
cells in order that causal inferences can be made on the role
of these neural circuit elements to behavior.
An array of techniques have been developed to activate specific cells in a spatially restricted manner but the use of cagedchemical compounds such as MNI-glutamate [142] or CDNIGABA [143] in combination with two-photon laser scanning
microscopy (TPLSM) has been the most popular. Nevertheless,
these techniques have inherent limitations including the lack of
spatial restriction for single cell activation due to diffusion of
the un-caged compounds, temporal imprecision in controlling
neuronal firing rates, certainly not suitable for in-vivo applications, and the inability to be generalized for stimulation of
different cell-types in the nervous system [144], [145].
Recent research endeavors were aimed at overcoming these
limitations by combining optogenetics and two-photon excitation to manipulate activities in single or multiple genetically
and spatially-targeted cells with high temporal resolution over
sustained intervals and within intact tissue volumes. The main
challenge in such efforts has been the need for the simultaneous opening of multiple opsins located throughout the
cell to overcome the modest conductance of individual lightgated channels (even the more light-sensitive opsin ChR2H134R). Rickagauer and colleagues were able to overcome
this obstacle and produce action potentials in cultured neurons
by introducing a complex spiral scan pattern to open sufficient
number of channels on individual neurons [146]. In two other
studies two-photon optogenetic stimulation were performed
in slice preparations but relied on development of innovative
hardware and using larger focal spots of laser illumination
[147], [148]. While the latter study yielded effective stimulation of single cells, the amount of laser power required
to generate action potentials was quite high (>100mW).
Consequently this technique is vulnerable to light scattering
events which potentially turns to a problem in applications
where deep tissue activation is necessary. A recent study has
shown activation of neurons more than 200 micron below
the surface using temporal focusing in combination with
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generalized-phase contrast [149]. While promising, the high
optical power per cell requirement still remains as the main
limitation specifically in applications where the activities of
multiple cells are controlled simultaneously.
The advent of new opsins, particularly the chimeric redshifted opsin C1V1 family, has allowed alternative methods
to be investigated. Prakash and colleagues took advantage of
the increased off-kinetics and the high maximal photocurrents
of C1V1 (compared to ChR2-H134R) to generate robust
sequence of action potentials with TPLSM in cultured neurons,
brain slice, and in-vivo preparations, Figure 9. The total power
required to stimulate an individual cell in this technique is
about 20mW where the single action potentials were produced
10-15ms after the initiation of a scan [150]. Moreover, they
observed that sub-cellular compartments of a neuron in slice
preparation could be activated using the same raster scanning
paradigm over the body of a single dendritic spine. Using
the same strategy, Packer and colleagues demonstrated that
using a spatial light modulator (SLM) in combination with
TPLSM C1V1 activation allowed multiple single cells to
be activated simultaneously [151]. Two-photon optogenetic
inhibition protocols were also developed for the first time by
using inhibitory opsins with increased photocurrent and longer
off-kinetics [150].
In order to effectively dissect the circuit elements that are
significant for neural computation and behavior, it will be
important to identify activity patterns within in-tact tissue.
Readouts for neural activity have been developed including
activity dependent calcium and voltage indicators. To extend
upon the power of two-photon optogenetic applications, a noninvasive all optical method to monitor induced or intrinsic
neural activity in parallel is necessary. Two important challenges for these experiments are: First, wavelengths should be
chosen precisely to avoid activating optogenetic tools while
reading the signal of activity-dependent probes (e.g., voltage
sensitive dyes or calcium indicators) and vice versa. This challenge can be addressed via development of spectral variants
of optical readout probes and two-photon opsins. Secondly,
spatiotemporal patterning of readout and manipulation must be
done simultaneously in the same intact volumes with arbitrary
patterns and at high speeds.
Arbitrary readout and manipulation requires multiple lasers
to be coupled to patterning modalities such as SLMs while
maintaining the galvanometer based scanning systems for activating, inhibiting and imaging neural populations. In terms of
speed, multiple strategies can be taken to improve the average
time to spike seen with two-photon optogenetic activation of
neurons. These include improvement in the scan trajectories
and developing more sensitive two-photon opsins. As noted in
[150], the typical raster scanning paradigm has an extensive
”fly-back” time where the galvanometers are repositioning to
the first pixel of the next line (v4ms on and v10ms off
per scan). By decreasing the unnecessary fly-back time, the
average time to spike can be decreased significantly. Moreover,
by engineering more sensitive two-photon opsins, scan times,
and thus time to spike initiation, can be decreased.
The non-invasiveness of two-photon optogenetic activation
of neurons allows for the development of algorithms for high-
16
throughput mapping techniques in ex-vivo and in-vivo preparations. It is known that the local connectivity maps of a neuron
depends not only on the type of cell, but also on where it
projects [152], [153]. There is evidence that local connectivity
between neurons in different regions of the brain are drastically
changed in some neurological diseases including autism [154].
Therefore, the high throughput mapping of neural connectivity
via two-photon optogenetics in intact tissue potentially opens
a new horizon in the study of such diseases.
Two-photon optogenetic stimulation combined with other
technologies will help to causally understand the fundamental
underpinnings of neural computation and behavior. By recreating neural activity patterns and using both cellular activity and
behavioral readouts, the sufficiency can be shown. Meanwhile,
by using the same readouts, and inhibiting the same patterns
of activity, the necessity can be shown.
Understanding details of the brain’s complex patterns of
activities that occur within milliseconds and across centimeters
of spatial distribution is a formidable task. The traditional
mechanisms for optogenetic stimulation, for example by implanting optical fibers within the brain tissue, do not have
the capability to elucidate how these micro circuits with a
diversity of elements and intricate topologies lead to a myriad
of behaviors since several hundreds to thousands of neurons
are controlled in a synchronous manner with each light pulse.
To causally dissect the spatiotemporal patterns of activity
necessary to study the behavior, each circuit element must be
controlled independently on the same time and length scales
as the brain functions. With the advent of simple and scalable
methods for two-photon optogenetic control over individual
neurons, we are approaching such goals as we are now able
to modulate multiple neurons at the resolution of single cells
with millisecond temporal precision.
VII. O PTOGENETIC M ICRO -EC O G; E XAMPLE OF A
H YBRID B RAIN I NTERFACE P LATFORM
A versatile brain interface should be able to control and
monitor brain activity across the large-scale networks of the
brain. Neural interfaces incorporating both electrode arrays
and optogenetic stimulation that reside on the surface of
the brain have the potential to fill a niche that exists between intracortical (e.g., optrodes) and minimally invasive
approaches (e.g., ofMRI). Optogenetics and electrode array
recordings offer millisecond temporal resolution and the ability
to spatially modulate and map neural sources.
Over the last few decades, several enabling technologies
have been developed to manipulate and record the brain’s
spontaneous and stimulus evoked activities, and each technique has both strengths and limitations. For instance, fMRI
has become a method of choice for in-vivo neurophysiological
analysis of brain function and has been translated into a
powerful clinical tool for diagnosis and treatment planning
for patients with brain tumors and other focal pathologies.
However, the ambiguity of interpreting recorded BOLD signals
plus its low temporal resolution are the main limitations
of fMRI. For stimulation, transcranial magnetic stimulation
(TMS) is noninvasive but offers very low spatial resolution
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Fig. 10.
Microfabricated electricorticography array fabricated on a transparent substrate and implanted under a cranial window for simultaneous
electrophysiological recordings and optogenetic stimulation. (a) A mouse sized array with connector. A grid of 16 platinum sites (150 micron diameter)
were fabricated with 500 micron site-to-site spacing and a nominal 1kHz impedance of 50kOhms. The arrays are implanted onto the dura, under a glass
cranial window. (b) A cross-sectional illustration of an implanted array with a photostimulus pattern created with a spatial light modulator is depicted. (c) A
brightfield image through a window shows the array resting atop the cortex in a chronically implanted mouse.
and electrical stimulation systems provide no strategy to target
specific cell types or to selectivty inhibit and excite cell
activity. Given the spatial, temporal and cell type advantages
afforded, hybrid interfaces incorporating optogenetics should
be explored. An example of such a system is the recently introduced optogenetic micro-electrocorticography (micro-ECoG)
platform.
ECoG arrays are implanted on the surface of the cortex
without penetrating into the brain. Compared to electroencephalography (EEG), they offer better spatial resolution and
signal to noise ratio [155]. ECoG was first implemented with
a set of discrete electrodes to map cortical function and
to localize aberrant activity in patients with epilepsy [156].
Over the next half century, large electrode arrays became
common clinical technology. In parallel, during the most
recent decade, ECoG based brain-computer interfaces (BCIs)
were implemented [159], [160], [161], and photolithography
was leveraged to create high-density microfabricated ECoG
(micro-ECoG) arrays [91], [89], [158]. Micro-ECoG arrays are
generally used for electrophysiological recordings, but towards
implementing bidirectional neural interfaces, the arrays could
be used for electrical microstimulation as well. However,
suppressing electrical stimulation artifacts in recorded signals
remains significant challenge [162], [163]. Considering all
benefits of optogenetics as summarized earlier, optical stimulation is potentially an ideal technique to be combined with
ECoG recording to create a new generation of optoelectronic
BCI instrumentation.
To create a bidirectional chronic neural interface, microECoG arrays were fabricated on a transparent biocompatible
polymer substrate and implanted over the dura, but under a
cranial window, in Thy1::ChR2-H134R mice [164], Figure 10.
Next, a spatial light modulator and fluorescence imaging system were employed to precisely pattern light onto the cortex
through the window and electrode array to stimulate or inhibit
neural activities. The micro-ECoG arrays were fabricated by
patterning platinum electrode sites on a transparent insulative
substrate polymer, Parylene C [164] which remains transparent
in the visible and infrared range of spectrum (400nm-1000nm)
[165] in contrast to some other commonly used materials in
flexible electrode array fabrication such as polyimide. Parylene
C, because of its biocompatibility [166], [167], is a wellknown and widely used polymer in neuroengineering particularly in fabrication of flexible implants [168], [169], [170].
The platinum electrode sites and connecting traces occluded
a small fraction of the device’s area, so optical access to the
cortex was maintained through the array [164].
Arrays composed entirely of transparent conductive polymers are potentially the next step in optogenetic micro-ECoG
technology. In-vivo imaging and photostimulus patterning directly under electrode sites would be possible in completely
transparent arrays, and fully polymeric fabrication could increase the flexibility and biocompatibility of the devices while
reducing production costs. Indium tin oxide (ITO), a transparent metal layer commonly used for LEDs and photovoltaic
cells, were used with PEDOT, a mostly transparent conductive
polymer, to make early transparent arrays [171]. However,
ITO is brittle. Laminating with PEDOT could bridge potential
cracks in the ITO, but forgoing metal all together may be just
as good. Conductive polymers can be patterned in multiple
ways including spin coating and lift-off, inkjet printing [172],
and many can be electropolymerized from their base monomer.
Electropolymerization is generally the most durable, but the
process requires an existing electrode. Maintaining mechanical
durability and electrophysiological recording quality similar to
that obtained with noble metals is an ongoing challenge as a
wider pallet of transparent materials are used to push a hybrid
of optogenetic and electrode array interfaces forward.
A. Source Localization and Intracortical Stimulation
One major application of optogenetic micro-ECoG is neural
source localization. Several algorithms exist to solve the
inverse problem of localizing neural activity from electrode
recordings, but testing and comparing the accuracy of these
algorithms is challenging using the existing methods [173],
[174], [175]. In most algorithms, the number of neural sources
and their spatial extent is set and ideally such assumptions
are testable. However, current electrical stimulation methods
(e.g., microstimulation or TMS) cannot easily create multiple sources at known locations or replicate diffuse activity.
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Fig. 11. Mapping of optogenetically induced cortical potentials. (a) Optogenetically evoked cortical potentials driven with a spatial light modulator (SLM)
and recorded by a micro-electrocorticography (micro-ECoG) array (4x4 grid of platinum sites, 500 micron site-to-site spacing, 150 micron site diameter,
50 kOhms @ 1 kHz) implanted under a cranial window in a Thy1::ChR2-H134R mouse. Spatial and temporal patterns of light were defined with digital
micromirror device (DMD) and projected onto the brain, through the cranial window and the array’s transparent substrate. In this example, two areas were
illuminated simultaneously (8ms, 4 mW/mm2 , 445nm, 60 trials averaged). Potentials nearest each photostimulus region had the greatest amplitude. Potentials
were recorded at 3 kHZ using a high-impedance amplifier (Tucker-Davis Technologies). The photostimulus was imaged through 400-500nm emission filter
with an EMCCD camera, (b) Potentials recorded on the cortical surface with micro-ECoG in response to photostimulation at three depths within the cortex
with a fiber-coupled laser. A micro-ECoG array was implanted epidurally in a Thy1::ChR2-H134R mouse. A laser-coupled fiber (200 micron, 0.2 NA, 2.4mW,
473nm) was first used to photostimulate at the cortical surface and then stereotactically inserted into the cortex 0.30 and then 0.60mm deep (50 trials at each
location).
Optogenetics, together with micro-ECoG and spatial light
modulation, creates a platform to emperically test source
localization methods over a range of spatially and temporally
defined neural sources in experimental models. Using this
platform, it is possible to evoke and record localized neural
activity by projecting patterns of light onto the cortical surface
while simultaneously mapping cortical potentials with a microECoG array, Figure 11(a). With the added precision of a DMD
projection system, one can aim around the electrode sites to
avoid any potential photoelectric artifact. It is also possible
to combine the optogenetic micro-ECoG technology with
spatial light modulators to produce a sequence of predesigned
illumination patterns where the location, area, and distance
separating sources are systematically varied to generate a large
dataset of micro-ECoG recording and neural activity caused
at known locations via optogenetic stimulation. This database
could be used to analyze complex signals and identify the
corresponding sources that are producing the recorded local
filed potentials.
Causing and recording neural activity at defined depths below the cortical surface would further improve understanding
of the ECoG signal and source localization methods. The
DMD projection system enabled precise photostimulation at
the cortical system. Holography [71], 2-photon methods [150],
and waveguides provide potential methods to photostimulate
within the cortex, under an implanted array. Figure 11(b)
shows cortical potentials evoked by photostimulating at three
depths within the cortex with a stereotaxically positioned fiber.
The relative amplitude of potential peaks following the stim-
ulus varried with fiber depth, but the largest potentials were
still colocalized with the fiber location. Similar experiments
with other opsins expressed under different promotoers could
fill out a cell type-specific study of ECoG.
B. In-Vivo Imaging with Optogenetic Micro-ECoG
Several in-vivo optical microscopy methods could be applied through the cranial window and transparent electrode
to image neurovascular, metabolic, and neural activity signals. Neurovascular coupling, the causal relationship between
neural activity and blood flow, is of great interest given
the importance of fMRI and the prevalence of neurovascular
diseases. Considerable progress has been made using sensory
stimulation [177], and more spatially defined cell type-specific
studies could be achieved with direct activation of neurons
through optogenetics. Brightfield images of a branch of the
middle cerebral artery (MCA) showed a large increase in
diameter following photostimulation, Figure 12. Simultaneous
potential recordings would help quantify the amplitude of optogenetically evoked activity and complete the neurovascular
picture. Further, intrinsic metabolic markers such as reduced
nicotinamide adenine dinucleotide (NADH) and flavin adenine
dinucleotide (FAD) could be imaged following optogenetic
stimulation, similar to studies using electrical microstimulation
and caged glutamate [178]. Miniaturized microscopes [179],
optical coherence tomography, and two-photon microscopy are
among the methods that could be used in conjunction with
optogenetics and microarrays to elucidate the neurovascular
system.
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Fig. 12. Diameter of the MCA increased by 20um following optogenetic
stimulation. Images were taken in sequence (a) before, (b) during, and (c)
after application of a train of blue pulses (133pulses/2s, 5ms, 9mW/mm2).
VIII. O PTOGENETIC A PPLICATIONS TO N EUROLOGICAL
AND P SYCHIATRIC D ISEASE
A. Overview
In the preceding pages, the protein engineering and technical
advancements that have made optogenetics such a promising
tool have been developed. Ultimately, our goal is to utilize opto-technology to map the nervous system and relieve
human suffering. To that end, optogenetics heralds a new
era of diagnostic and therapeutic approaches to neurological
and psychiatric disorders. Its power to unwrap disease states
is multi-fold, and begins first by the ability to selectively
perturb and interrogate brain nuclei and circuits in order to
determine elements critical to normal function. The second is
to determine points at which intervention is possible. Finally,
new therapeutic avenues are unlocked through the unique
power of optogenetics as a tool to selectively control cell
types and circuits. A comprehensive summary of the current
state of the field is beyond the scope of this review, and
the reader is directed to other reviews on this topic [8], [9],
[63], [180], [181], [182]. We focus here on the approach to
considering diseases of the nervous system as dysfunction of
neural circuits, and then novel ways to combine optogenetics
with temporal circuit dynamics to achieve improved knowledge of pathophysiological mechanisms and opportunities for
therapeutic interventions. Finally, we conclude by imagining
the (near) future in which optogenetic techniques are combined
with other powerful tools for disease analysis and therapeutics,
to realize its full potential within this realm.
Neurological and psychiatric diseases of the brain were traditionally understood as loss or perturbation of function based
on changes in cell mass (neurodegenerative diseases), cell
function (loss of memory), neurotransmitter systems (Parkinson’s), or disrupted connections (addiction). Unfortunately,
most disorders result from a combination of such derangements, and conventional therapies are, by definition, either
too limited in their approach or too broad in their side-effect
19
profile. Furthermore, the finer structure of the cortical and
subcortical circuits that serve these input-output loops are incompletely understood, defined by genetically and chemically
heterogenous populations of neurons and their projections.
Finally, different disease states may share similar pathological
features, such as altered beta or gamma oscillations, but
produce distinct phenotypes, such as schizophrenia, autism and
Parkinson’s disease.
To understand the normal and disrupted function of circuits, optogenetics has been used to great effect to dissect
them at different levels in the brain. At the level of the
synapse, the functional communication point between neurons,
channelrhodopsins have been used to explore both synaptic
potentiation and depression [184], [185], [186], providing a
deeper understanding of circuit development and pathologies.
By stimulating both cell bodies and axonal projections, optical circuit mapping has been initiated to understand local
connectivity amongst various neuronal cell types [187], [188]
and long-range connectivity between brain regions [187],
[189]. Activation and inhibition of these connections locally
and at a distance may alter the balance of neuromodulation.
For instance, an imbalance in dopamine activity within the
indirect and direct pathways of the basal ganglia is thought
to underlie Parkinson’s disease. By targeting expression of
ChR2 to two different classes of dopaminergic neurons (D1,
D2), Kravitz and colleagues [190] were able to demonstrate
prokinetic and antikinetic pathways in the basal ganglia, by
direct photostimulation of each dopamine subtype in normal
animals. In an animal model of Parkinson’s (6-OHDA) they
were then able to rescue the disease phenotype by selectively
photostimulating D1-ChR2-expressing medium spiny neurons,
providing a potential therapeutic intervention point for deep
brain stimulation (DBS). The mix and distribution of local
neuronal connections also affects regional inhibition versus
excitation. Using targeted expression of the super step-function
opsin (SSFO) in both excitatory and inhibitory cells of the medial prefrontal cortex (mPFC), a region that exerts significant
top-down control over decision-making, impulse control, and
many social behaviors, the balance of excitation and inhibition
in this region of the brain could be selectively altered [9].
Mice were tested on a variety of social interaction tests and
in a fear-conditioning paradigm. Increasing the balance of
excitation over inhibition caused disturbed social interactions
and an inability to form appropriate memories for conditioned
responses, whereas increasing inhibition did not. Furthermore, these behavioral pathologies were associated with an
increase in gamma oscillations in the local field potentials
of mPFC neurons. These results are striking because a novel
optogenetic tool, SSFO, was utilized to define a mechanism
that might link disparate observations of changes in cortical
excitatory/inhibitory balance, relative loss or hypofunction of
mPFC inhibitory interneurons, altered gamma oscillations, and
social and cognitive dysfunction in diseases such as autism and
schizophrenia. Two complementary optogenetic studies have
provided a more precise link between the loss of inhibitory
cortical neurons and the aberrant generation of gamma oscillations in these diseases, potentially linking these to the
alterations in observed cognitive flexibility. In an in-vivo study,
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Cardin and colleagues [191] genetically targeted ChR2 to
either inhibitory interneurons or excitatory pyramidal cells of
the somatosensory barrel cortex. These populations were then
selectively photostimulated over a range of frequencies. Only
stimulation of inhibitory (but not excitatory) neurons at gamma
frequencies generated local field potentials in the gamma
frequency range. Further, they demonstrated that inhibitory
interneuron-induced gamma rhythms temporally sharpened the
transmission of sensory information from the periphery. In an
in-vitro study utilizing bidirectional optogenetic control in a
brain slice preparation from PFC, suppression of interneurons was found to dampen local gamma band power while
feedback inhibition of interneurons on downstream excitatory
cells generated emergent gamma oscillations, demonstrating a
necessary and sufficient role for these inhibitory cells in the
generation of cortical gamma rhythms [16]. Evoked gamma
oscillations also increased the gain of the neuronal response
to realistic spike trains by increasing mutual information and
decreasing noise entropy. Both studies have provided the first
support for a causal role for interneurons in the generation
of gamma, and their role in sharpening cortical sensory
transmission and enhancing mutual information in connected
cells. These results imply that loss or aberrant expression of
inhibitory cortical cells, often observed in psychiatric disease,
may lead to the cognitive derangements seen in patients with
schizophrenia and autism.
Optogenetics combining circuit manipulations and behavioral phenotypes has also been used to great success to
determine projection-specific targets within normal function
and disease. One very successful example is in defining the
cortico-brainstem circuitry underlying depressive phenotypes.
Most interventions (pharmacological, electroconvulsive therapy, light therapy, transcranial magnetic stimulation) have had
incomplete success and multiple side-effects, partly due to
the lack of specificity of these treatments and an incomplete understanding of the appropriate circuit sites to intervene. Pharmacological approaches have been particularly nonspecific, often globally antagonizing serotogenergic signaling
systems in the brain, producing unwanted effects. Just as
with some of the disorders of social dysfunction described
earlier, the medial pre-frontal cortex (mPFC) sends multiple
projections to subcortical nuclei implicated in depression. In
an elegant demonstration of the power of optogenetics to query
projection specificity, Warden and colleagues [192] optically
defined two heterogenous circuits that alternately lead to
impulse control disorders and amotivational states, initiated
in mPFC. Typically, depressed patients express a degree of
hopelessness when challenged with difficult situations. Using
a test of learned helplessness in rats challenged with a water
environment, selective photostimulation of one set of mPFC
projections, terminating in the brainstem dorsal raphe nucleus
(DRN), reversibly rescued the depressive phenotype. However,
photostimulation of an alternate projection from mPFC to
the lateral habenula increased immobility, mimicking more
strongly the depressive phenotype. Surprisingly, global stimulation of mPFC primary cortical projection neurons had no
effect on behavior. These studies provided a means to dissect
depressive circuitry, and identify a point of intervention for
20
future applications of deep brain stimulation (DBS), taking
advantage of this cell-type and projection specificity to limit
some of the side effects associated with non-selective electrical
stimulation. A similar optogenetic strategy was used to define
opposing roles for distinct subregions of the bed nucleus
of the stria terminalis (BNST) in anxiety behaviors, both
groups of neurons having multiple, distinct projections which
independently control individual features of the anxiogenic and
anxiolytic response [193].
Optogenetics has been used in this projection specific manner to disentangle the role of brain regions that participate
in varied activities. One example is the amygdala, a heterogeneously dense collection of cell types that participates in
diverse up- and downstream connections. The amygdala is
a deep brain structure implicated in processing and forming
memories for emotionally salient or fear-laden experiences.
In one set of experiments, by combining projection specificity of opsin expression with directionally-restricted illumination techniques, a causal role for the basolateral to central
amygdalar circuit in promoting anxiety was defined [183].
Alternately, a role for the basolateral amygdalar projection
to the nucleus accumbens, another deep brain nucleus, was
demonstrated for reward seeking and self-stimulation [182].
Though both behaviors can be linked to the development
of substance abuse, they are quite distinct, and originate
from within the same nucleus, differentiated by their discrete
projection patterns.
Combining optogenetics with behavioral read-out methods
can also be used to determine up- and downstream effectors of
nuclei within a circuit, and the relationship between patterns
of activity within these nuclei and behavioral phenotype.
One of the first such demonstrations was in the arousal
circuit, a series of interconnected subcortical nuclei. Understanding the arousal system is important because disorders
of sleep (e.g. narcolepsy) and consciousness (e.g. delayed
emergence from anesthesia, coma) arise from derangements
in these pathways. Within this pathway, a group of neurons
of the lateral hypothalamus (LH) releases peptides known
as hypocretins/orexins [194], [195]. Gene deletions in the
hypocretins lead to disorders such as narcolepsy [196], and
activity and downstream signaling in hypocretin neurons had
been correlated but not causually linked to wakefulness.
Adamantidis and colleagues [197] achieved this by leveraging
a lentivirus strategy to express ChR2 in LH. Direct stimulation
of hypocretin-producing neurons within this nucleus increased
the probability of sleep to wake transitions, and shortened the
latency to wakefulness in a rate-dependent fashion. Until this
experiment was performed, it was not known that release of
these neuropeptides was sufficient to promote the transition to
wakefulness. Stimulation rates of <5Hz produced no effect
while those between 5-20Hz shifted latency, demonstrating
that different patterns of activity within the same circuit could
have completely different behavioral effects. A similar causal
role for norepinephrine in arousal was demonstrated by using
a Cre-driver line to express ChR2 in the locus coeruleus
(LC) [198]. Precise optical activation of LC neurons caused
sleep to wake transitions with a shorter time constant than
for hypocretin neurons. Using combined photo-modulation of
IEEE REVIEWS IN BIOMEDICAL ENGINEERING, DRAFT-REVISED
LH and LC in sequence, they then showed that LC is both a
necessary and sufficient downstream effector of the wakefulpromoting effects of the hypocretins. These studies should
enable new circuit-specific and rate-dependent targets for the
treatment of narcolepsy, other disorders of sleep and arousal,
and delayed emergence from general anesthesia.
B. The optogenetic revolution in disease
The range of circuit-specific derangements in neurological
disease is vast and complex, and optogenetic tools have
provided new methods to break open these circuit codes. However, the reach of optogenetics in diagnostics and therapeutics
requires more imagination. Utilizing animal models of disease,
optogenetic techniques can be applied to treating these circuits.
One such mechanism is by applying modulated patterns of
activity. For instance, the selective photoinhibition of cortical
excitatory cells [199] or ventrobasilar thalamic cells [200] by
halorhodopsin can abort seizure-like bursting activity in-vivo
and in-vitro models of epilepsy. Studies using optogenetics
have also been used to define specific nuclei, projections, and
rate codes for the therapeutic effect of DBS in Parkinson’s
disease [15], [190] and depression [201], [203], minimizing
side-effects or the cancelation of opposing effects that can be
caused by electrically stimulating brain regions with a mixture
of diverse cell types. More generally, there is great hope that
optogenetics will provide the ability to treat brain diseases that
in the past, were ameliorated by surgical removal or lesioning,
through a more precise functional resection. Optomodulation
of these circuits is certainly a less invasive means of achieving
therapeutic effects. Pattern-specific optomodulation could also
be used to correct aberrant oscillatory patterns that are the
basis of so many diseases [16], [191].
Another possible role for optogenetic therapeutic interventions is to restore or probe newly functional connections.
Multiple techniques might exist to achieve this end. As the
adage ”neurons that fire together, wire together” (the Hebbian
rule of plasticity [202]) states, driving pre-synaptic terminals,
either to restore transmitter release, or in a temporally precise
fashion to strengthen synaptic connections, could re-establish
synapses that have been lost (e.g. in neurodegenerative diseases), or engineer synapses that do not currently exist (e.g.
to enhance memory or other function). The restoration of
functional connections might also be achieved by combining
light-sensitive technologies with brain-machine interfaces, to
create closed loop systems in which either optical or electrical
sensors are used to read circuit activity and then respond with
either optical or electrical actuators [199], [200]. Finally, in
therapeutic models in which stem cells have been grafted into
a host system, optogenetics can be used to assess synaptic to
circuit level wiring. Tonnesen and colleagues [204] describe
utilizing optogenetic techniques to test synaptic connectivity
following dopamine stem-cell replacement in an animal model
of Parkinson’s disease.
Optogenetics might not only be used to test therapies,
but to generate reversible animal models for disease [205].
Such models might be created by introducing opsins (through
targeted viral delivery or the generation of transgenic lines)
21
into a number of potential locations within a circuit, and
then activating or inhibiting that circuit with precise temporal
dynamics. In this paradigm, dysfunction is induced with light,
while multiple pharmacological, electrical or surgical interventions to ameliorate symptoms might then be tried with high
throughput in order to determine the most effective therapy.
The optogenetic toolkit has now expanded from the traditional light-gated ion channels, which activate or silence
neuronal activity, to opsins that activate intracellular events.
This is important as many of the targets of disease are intracellular, and transcriptional and epigenetic events govern the
expression of many neuropsychiatric disorders. A new array
of photoswitchable molecules that activate G protein-coupled
signaling, nucleotidyl cyclases, glutamate receptor signaling,
and intracellular trafficking have created the opportunity to
use the precise delivery of light to control downstream events
[207], [208], [209], [210], [211], [50]. In some cases, lightgated enzymes and transcription factors can be combined
to achieve selective gene expression or control inputs and
behavior in intact animal [51], [212], [213], [214], [215],
[216], [217], [218], [219], [220]. Ultimately, such manipulations affect can affect transcription and epigenetic expression,
leading to permanent cell, circuit, and heritable behavioral
changes. Immediate early genes such as c-fos, Arc, Npas4
and Zif268 [197], [221], and their upstream transcriptional
regulatory elements [222], have been used in combination with
photostimulation to identify cells that have been differentially
activated or silenced during optomodulation. Together, optical
stimulation combined with mapping gene expression changes
in up- or downstream elements opens new doors to linking
behavior to genetic changes. Novel approaches in which highthroughput techniques to characterize the mRNA transcribed
in response to optogenetically-controlled behaviors or circuit
activation (”integrated optogenetic-transcriptomics approach”)
have been described in detail elsewhere [182].
C. Final challenges - human therapeutic applications
While optogenetics has shown great promise in circuitbreaking neuropsychiatric diseases and finding points of intervention within animal models, human therapeutics has proven
more elusive. For one, there is considerable light-scattering in
brain tissue, with blue wavelengths (473 nm) used to activate
ChR2 diminishing to v1% of their initial power 1mm away
from the illumination source [9]. As brain volume scales from
rodent to non-human primate to human by a factor of at least
×103 , the potential stimulated volume in the human brain is
cut drastically [223]. Indeed, although there has been some
success achieving opsin expression and activation in primate
cortex, optogenetically-controlled behaviors are more difficult
to achieve than they have been in rodents [34], [61]. This may
be related to sparse volumetric photo-excitation of primate
cortex. Only a few primate studies, thus far, have achieved
optogenetic control of behavior, and these have all been in the
visual system [116], [224], [225]. In general, volumetric recruitment of excitable brain tissue has been achieved by using
tricks such as pan-cellular promoters (CAG) for expression,
task-based fMRI navigation to direct viral injections, and dual
IEEE REVIEWS IN BIOMEDICAL ENGINEERING, DRAFT-REVISED
electrodes to achieve greater volumetric excitation. Thus, more
precise brain-behavior targeting strategies, more widespread
infection, larger volumetric stimulation, or transduction with
opsins with higher photon sensitivity or longer wavelength
absorption for deeper light penetration will be required to
achieve tissue activation in primates.
It may be that the typical optical stimulation will have
to be complemented by even newer non-photostimulating
techniques [226], or methods that utilize an optical window
(e.g. transparent silicone artificial dura) to guide injections
and track expression in such a way that the underlying cortex
is not excessively damaged or deformed [228]. Such an
optical window may also permit bulk volume illumination of
superficial layers of cortex. The second is genetic specificity
and targeting strategies unique to non-human primates and humans. Tropism, vector packaging, and cross-species variations
in promoter specificity are limitations requiring new testing.
Further, the efficiency of gene expression in non-human primates and humans is currently a poorly defined variable. Early
CNS gene therapy reveals successes with both lentiviral and
AAV transduction, though AAV may be the more promising
vector secondary to decreased immunogenicity [229]. Unfortunately, different serotypes of AAV do not uniformly transduce
heterologous brain regions, and this suggests that expression
efficiency will have to be tested on a serotype and region-byregion basis. The volumetric light sensitivity of a given volume
of brain will be related to expression efficiency of the opsin,
potentially in a non-linear fashion, but both acute and chronic
overexpression might also be neurotoxic. In some of the first
studies in which opsins were expressed in macaque cortex,
injections of opsins packaged within AAV5 [61] or lentivirus
[34] vectors under control of hSyn- and hThy-1 or α-CaMKII
led to expression efficiencies of 60-80% within 1mm of the
site of injection (viral titer of 109 -101 0 particles/ml) after 45 months. Longitudinal studies in non-human primates are
crucial to determining the duration of stable opsin expression,
the duration of maximal recruitment and light sensitivity, and
the cross-over point into neurotoxicity.
In order to enable the evolution of delivery technologies,
new optogenetic toolkits are being developed in non-human
primates [34], [61], [227], [228]. In the case of viral gene
delivery, human safety is also an issue. The AAV family is less
pathogenic and immunogenic than lentiviruses, decreasing the
probability of host cell mutagenesis after infection. Invasive
techniques for modulation are also less desirable than noninvasive techniques for human photomodulation. Delivery of
drugs and/or viruses across the blood-brain barrier (BBB) is
typically difficult. One option for gene delivery might be direct
injection into the brain parenchyma or ventricles/cisterna to
circumvent such difficulties [230]. There is recent evidence
that some serotypes of AAV may cross the BBB, and could
therefore be delivered via intravenous injection [231], [232].
Newer, less invasive technologies to deliver small designer
drugs or molecules have been proposed using conjugated
nanoparticles or specific enzyme-substrate pairs, cleaved after
crossing the BBB [233], [234], [235], [182]. In addition,
toolboxes of cellular and regional mini-promoters in the brain
are being defined [236] that can be utilized for targeting
22
applications. Ultimately, optogenetics may exploit genetics,
not simply for targeting cell types for expression, but to create
personalized toolkits to treat patients with a particular DNA
makeup, utilizing opsin therapeutics aimed at their genotype,
not phenotype.
IX. C ONCLUSION
Optogenetics, as a new methodology for neurostimulation,
has become a popular technique that is widely used in the
study of the nervous systems in different animal models.
The specific cell-type targeting capability of optogenetics has
opened new horizons in dissecting the circuitry of neurological
disorders and the parallelism of optics has made it possible to
study the dynamics of large-scale neural networks particularly
in the cortex. The advent of optogenetics has opened new
lines of research including the development of new lightsensitive proteins, the realization of technologies for light
delivery and detection in brain tissue, the study of diseases,
and the combination of optogenetics with other established
brain stimulation and recording systems such as electrode
arrays and magnetic resonance imaging.
Optogenetics has also opened a new line of thinking among
neuroscience and bioengineering communities. For instance,
one question is whether it is possible to use other forms of
energy, instead of light, to manipulate neural activities. Using
acoustic waves instead of light can potentially help to noninvasively reach deeper inside the tissue with the cost of
reducing the spatial resolution. The tools of optogenetics were
originally adopted from nature. Further searching in the nature
to find new forms of energy-gated ion channels is potentially
a new research endeavor for next few years which can benefit
from recent advances in biotechnology and bioinformatics.
Extending the scope of optogenetics may be possible though
the development of other light sensitive proteins (e.g., enzymes
that are dysfunctional until exposed to light).
The success story of optogenetics particularly highlights
the importance and value of basic-science research. Perhaps,
the scientists who were studying the metabolism of algae
and bacteria few decades ago could not predict that their
discoveries will eventually help to develop one of the most
powerful tools for the study of the brain at least in early the
21st century.
ACKNOWLEDGMENT
Justin Williams, Ramin Pashaie, and Thomas Richner’s
research was sponsored by the Defense Advanced Research
Projects Agency (DARPA) MTO under the auspices of Dr.
Jack Judy through the Space and Naval Warfare Systems
Center, Pacific Grant/Contract No. N66001-12-C-4025. Ramin
Pashaie would like to also appreciate the University of Wisconsin research growth initiative grants# 101X172, 101X213,
and 101X254.
Polina Anikeeva’s research is sponsored by National Science Foundation (NSF, MRSEC DMR-0819762 and NSF
CAREER CBET-1253890) and by the Defense Advanced
Research Projects Agency (DARPA YFA D13AP00045).
IEEE REVIEWS IN BIOMEDICAL ENGINEERING, DRAFT-REVISED
Jin Hyung Lee would like to acknowledge her research support provided by the NIH/NIBIB R00 Award
(4R00EB008738), Okawa Foundation Research Grant Award,
NIH Directors New Innovator Award (1DP2OD007265), the
NSF CAREER Award (1056008), and the Alfred P. Sloan
Research Fellowship.
Ofer Yizhar is supported by the Human Frontier Science Program under grant# 1351/12 from the Israeli Science
Foundation and the Israeli Center of Research Excellence in
Cognition grant# I-CORE Program 51/11. Matthias Prigge
is supported by a postdoctoral fellowship from the Minerva
Foundation.
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IEEE REVIEWS IN BIOMEDICAL ENGINEERING, DRAFT-REVISED
Ramin Pashaie received the B.S. degree in electrical engineering (with major in microelectronics)
from Shahid Beheshti University (SBU), Tehran,
Iran, in 1998, the M.S. degree in fields and waves
from Khaje Nasir Tousi University of Technology
(KNTU), Tehran, Iran, in 2001, and Ph.D. degree in
electrical and systems engineering from University
of Pennsylvania, Philadelphia, in Dec 2007 under supervision of Professor Nabil H. Farhat. After Ph.D.,
he joined Karl A. Deisseroth lab as a postdoctoral
scholar in the bioengineering department at Stanford University. During his postdoctoral training he focused on technology
development for optical modulation of neural activities using the tools of
photonics and molecular genetics. From September 2009 he joined the
electrical engineering department at University of Wisconsin-Milwaukee as an
assistant professor and director of the Bio-Inspired Sciences and Technology
Laboratory (BIST-LAB) where the research is about optical interrogation of
the dynamics of large scale neural networks mostly in the brain cortical
regions. In particular, he is currently interested in implementation of neuroprosthetic devices to extract details of information processing in cortical
networks and the nonlinear dynamics of cortical columns. This information
can be used for reverse engineering and realization of brain machine interface
mechanisms.
Polina Anikeeva received her BS in Physics from
St. Petersburg State Polytechnic University in 2003.
After graduation she spent a year at Los Alamos
National Lab where she worked on developing novel
types of photovoltaic cells based on semiconductor
quantum dots. She then enrolled in a PhD program in
Materials Science at MIT and graduated in January
2009 with her thesis titled Physical Properties and
Design of Light Emitting Devices and Nanoparticles.
She completed her postdoctoral training at Stanford
University, where under supervision of Karl Deisseroth she developed implantable devices for simultaneous optical stimulation
and high-throughput electronic recording from neural circuits during free
behavior. Polina joined the faculty of the MIT Department of Materials
Science and Engineering in July 2011 as AMAX career development assistant
professor. Her lab at MIT focuses on the development of flexible and
minimally invasive materials and devices for neural recording, stimulation
and repair. She is also a recipient of Sanofi Biomedical Innovation Award,
NSF CAREER Award, DARPA Young Faculty Award and the Dresselhaus
Fund Award.
Jin Hyung Lee is an Assistant Professor of Bioengineering, Neurology and Neurological Sciences,
and Neurosurgery at Stanford University. Dr. Lee
received her Bachelor’s degree from Seoul National
University in 1998 and Masters in 2000 and Doctoral
degree in 2004 from Stanford University, all in
Electrical Engineering. She is a recipient of the
2008 NIH/NIBIB K99/R00 Pathway to Independence Award, the 2010 NIH Director’s New Innovator Award, the 2010 Okawa Foundation Research
Grant Award, the 2011 NSF CAREER Award, the
2012 Alfred P Sloan Research Fellowship, the 2012 Epilepsy Therapy Project
Award, and the 2013 Alzheimer’s Association New Investigator Award. She
pioneered the optogenetic functional magnetic resonance imaging technology
and as an Electrical Engineer by training with Neuroscience research interest,
her goal is to analyze, debug, and engineer the brain circuit through innovative
technology.
28
Rohit Prakash Rohit Prakash received his Bachelor
of Sciences degree in physics with a minor in philosophy from the University of North Carolina, Chapel
Hill in 2005. He received his Ph.D in Neuroscience
from Stanford University under the direction of
Karl Deisseroth in 2012. He is currently completing
his M.D./Ph.D. Rohit’s interest are within combining nanotechnology applications, optical engineering
techniques, and optogenetics in order to build tools
to probe the causal underpinnings of human behavior.
Ofer Yizhar received the B.S. degree in biology
from the Hebrew University in Jerusalem, Israel.
During his Ph.D. studies at the Tel Aviv University,
he studied the pre-synaptic mechanisms of neurotransmitter release using electrophysiological and
imaging techniques and developed software for analysis of single-vesicle motion in time-lapse movies.
Working in the Deisseroth Lab, Stanford University,
Stanford, CA, he has developed and applied new
optogenetic tools to the study of the neural circuits
involved in the pathophysiology of neuropsychiatric
disease. From 2011, Dr Yizhar has established his lab at the department of
Neurobiology in Weizmann Institute of Science where his research is focused
on combining molecular biology, electrophysiology, imaging and behavioral
techniques to study neural circuits.
Matthias Prigge After obtaining his diploma degree in biochemistry from the Free University in
Berlin in 2005, he became a research assistant in
the membrane biophysics group at Johannes Kepler
University in Austria. Finding his way back to
Berlin, he joined the group of Peter Hegemann at
Humboldt University and developed new channelrhodopsin variants for optogenetic research. Upon
finishing his PhD in biophysics in 2012, Dr. Matthias
Prigge joined the neuroscience group of Ofer Yizhar
at the Weizmann Institute of Science in Israel. Here
he is interested in engineering new light activatable biological tools to dissect
neuronal circuits in behaving animals.
Divya Chander completed her MD, PhD at the
University of California, San Diego and the Salk
Institute in the Systems Neurobiology Lab, studying
adaptive gain control in the retina. She did a residency in Anesthesia at the University of California,
San Francisco and a post-doctoral fellowship in
the Deisseroth lab at Stanford University. She is
currently on the faculty in the Department of Anesthesia at Stanford University School of Medicine.
Dr. Chander’s research interests focus on transitions
between levels of arousal, using natural sleep-wake
or anesthesia-induced unconsciousness as her model systems. She uses optogenetic approaches to dissect these circuits in rodents and translates these
insights to intraoperative EEG-based studies in humans undergoing changes
in levels of consciousness
IEEE REVIEWS IN BIOMEDICAL ENGINEERING, DRAFT-REVISED
Thomas J. Richner received a B.S. degree in
biomedical engineering from Washington University
in St. Louis in 2008, a B.S. degree in Mathematics
from Northland College in 2006, and a M.S. in
biomedical engineering in 2010 from the University
of Wisconsin-Madison where he is currently a PhD
candidate. His research interests include electrocorticography, optogenetics, and neural interfacing.
Justin Williams is currently the Vilas Distinguished
Achievement Professor in Biomedical Engineering and Neurological Surgery at the University of
Wisconsin-Madison. He received B.Sc. degrees in
mechanical engineering in 1995 and in engineering
physics in 1996 from South Dakota State University,
Brookings. He went on to the M.Sc. and the Ph.D.
degree in bioengineering in 2001 from Arizona State
University, Tempe. From 2002 to 2003, he was a
jointly appointed Research Fellow in the Department
of Biomedical Engineering, University of Michigan,
Ann Arbor, and a Research Scientist in the Department of Neurological
Surgery, University of Wisconsin, where his work focused on neurosurgical
applications of neural engineering devices. He joined the department of
Biomedical Engineering at the University of Wisconsin in 2003. His research
interests include BioMEMS, neuroprostheses, and functional neurosurgery.
29